Selective neuronal nitric oxide synthase inhibitors

ABSTRACT

Peptidomimetic compositions for selective inhibition of neuronal nitric oxide synthase.

This application claims priority benefit from pending U.S. provisionalpatent application Ser. No. 06/300,130 filed Jun. 22, 2001 and U.S.provisional patent application Ser. No. 60/315,587 filed Aug. 29, 2001,both of which are incorporated herein in their entirety.

The United States government has certain rights to this inventionpursuant to Grant No. GM49725 from the National Institutes of Health toNorthwestern University.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) is synthesized enzymatically from arginine in numeroustissues and cell types by a family of enzymes, collectively known asnitric oxide synthase (NOS, E.C. 1.14.13.39). Three principal isoformsof this enzyme have been isolated and characterized, each associatedwith different physiological functions: the immune response (inducibleNOS or iNOS), smooth muscle relaxation (endothelial NOS or eNOS), andneuronal signaling (neuronal NOS or nNOS). All of these isoforms utilizeNADPH, FAD, FMN, (6R)-5,6,7,8-tetrahydrobiopterin and heme as cofactors.

Overproduction of NO has been a factor in numerous disease states. NOoverproduction by nNOS has been implicated in strokes, migraineheadaches, Alzheimer's disease, and with tolerance to and dependence onmorphine. iNOS-mediated overproduction of NO has been associated withdevelopment of colitis, tissue damage and inflammation, and rheumatoidarthritis.

Animal studies and early clinical trials suggest that NOS inhibitorscould be therapeutic in many of these disorders; however, because of theimportance of nitric oxide to physiological functioning, potent as wellas isoform-selective inhibitors are essential. nNOS inhibition has beentargeted for treatment of strokes, and iNOS inhibition for the treatmentof septic shock and arthritis. Although there may be pathologiesassociated with overactivity of eNOS, blood pressure homeostasis is socritical that most investigators believe that therapeutically useful NOSinhibitors should not inhibit eNOS.

Excellent inhibitory potency and selectivity for nNOS over eNOS and iNOShave been achieved with certain prior art (FIG. 1) nitroargininedipeptide amides that have an amine-containing side chain (1-3). SeeHuang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R.B. N^(ω)-Nitroarginine-Containing Dipeptide Amides. Potent and HighlySelective Inhibitors of Neuronal Nitric Oxide Synthase. J. Med Chem.1999, 42, 3147-53.

The most potent nNOS inhibitor among these compounds isL-Arg^(NO2)-L-Dbu-NH₂ (1) (K_(i)=130 nM), which also shows excellentselectivity over eNOS (>1500-fold) and 192-fold selectivity over iNOS.Further, peptidomimetic modifications are, however, invariably necessarybefore such compounds can be therapeutically useful. Generally, peptideshave poor bioavailability and are generally unsuccessful drugcandidates.

The foregoing background information, together with other aspects of theprior art, is described more fully and better understood in light of thefollowing publications: (1) Kerwin, J. F., Jr.; Lancaster, J. R., Jr.Nitric Oxide; A New Paradigm for Second Messengers. Med. Res. Rev. 1994,14, 23-74; (2) Kerwin, J. F., Jr.; Heller, M. The Arginine-Nitric OxidePathway: A Target for New Drugs. J. Med. Chem. 1995, 38, 4342-62; (3)Stuehr, D. J.; Griffith, O. W. Mammalian Nitric Oxide Synthases. Adv.Enzymol. Relat. Areas Mol. Biol. 1992, 65, 287-346; (4) MacMicking, J.;Xie, Q. W.; Nathan, C. Nitric Oxide and Macrophage Function. Annu. Rev.Immunol. 1997, 15, 323-50; (5) Forstermann, U.; Pollock, J. S.; Schmidt,H. H. H. W.; Heller, M.; Murad, F. Calmodulin-DependentEndothelium-Derived Relaxing Factor/Nitric Oxide Synthase Activity isPresent in the Particulate and Cytosolic Fractions of Bovine AorticEndothelial Cells. Prot. Natl. Acad. Sci. U.S.A. 1991, 88, 1788-92; (6)Schmidt, H. H. H. W.; Walter, U. NO at Work. Cell 1994, 78, 919-25;(7)(a) Choi, D. W.; Rothman, S. M. The role of glutamate neurotoxicityin hypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 1990, 13,171-82; (b) Garthwaite, J. In the NMDA Receptor; Watkins, J. C.Collingridge, G. L., Eds.; Oxford University Press.; Oxford, England,1989; pp 187-205; (8) Thomson, L. L.; Iversen, H. K.; Lassen, L. H.;Olesen, J. The role of nitric oxide in the migrane pain. CNS Drugs 1994,2, 417-22; (9) Dorheim, M. A.; Tracey, W. R.; Pollock, J. S.; Grammas,P. Nitric Oxide synthase activity is elevated in brain microvessels inAlzheimer's disease. Biochem. Biophys. Res. Commun. 1994, 205, 659-65;(10) Bhargava, H. N. Attenuation of tolerance to, and physicaldependence on, morphine in the rat by inhibition of nitric oxidesynthase. Gen. Pharmacol. 1995, 26, 1049-53; (11) Seo, H. G.; Takata,I.; Nakamura, M.; Tatsumi, H.; Suzuki, K.; Fujii, J.; Taniguchi, N.Introduction of nitric oxide and concommittant suppression of superoxidedismutase in experimental colitis in rats. Arch. Biochem. Biophys. 1995,324, 41-7; (12) Kubes, P.; Suzuki, M.; Granger, D. N. Nitric Oxide; anendogeneous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci.U.S.A. 1991, 88, 4651-5; (13) Maclintyre, I.; Zaidi, M.; Towhidul Alam,A. S. M.; Datta, H. K.; Moonga, B. S.; Lidbury, P. S.; Hecker, M.; Vane,J. R. Osteoclastic inhibition; an action of nitric oxide not mediated bycyclic GMP. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2936-40; (14)Kilbourn, R. G.; Jubran, A.; Gross, S. S.; Griffith, O. W.; Levi, R.;Adams, J.; Lodato, R. F. Reversal of endotoxin-mediated shock byN^(G)-methyl-L-arginine, an inhibitor of nitric oxide synthesis.Biochem. Biophys. Res. Commun. 1990, 172, 1132-8; (15)(a) Collins, J.L.; Shearer, B. G.; Oplinger, J. A.; Lee, S.; Garvey, E. P.; Salter, M.;Duffy, C.; Burnette, T. C.; Furfine, E. S. N-Phenylamidines as selectiveinhibitors of human neuronal nitric oxide synthase. Structure-activitystudies and demonstration of in vivo activity. J. Med. Chem. 1998, 41.2858-71; (16) Wright, C. W.; Rees, D. D.; Moncada, S. Protective andPathological roles of nitric oxide in endotoxin shock. Cardiovasc. Res.1992, 26, 48-57; (17) Garvey, E. P.; Oplinger, J. A.; Furfine, E. S.;Kiff, R. J.; Laszlo, F.; Whittle, B. J. R.; Knowles, R. G. 1400W is aslow, tight binding, and highly selective inhibitor of inducible nitricoxide synthase in vitro and in vivo. J. Biol. Chem. 1997, 272,4959-63;(18) Huang, H; Martasek, P.; Roman, L. J.; Masters, B. S. S.;Silverman, R. B. N^(ω)-Nitroarginine-Containing Dipeptide Amides. Potentand Highly Selective Inhibitors of Neuronal Nitric Oxide Synthase. J.Med Chem. 1999, 42, 3147-53.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows several peptidomimetic compounds of the prior art.

FIG. 2 shows several of a class of compositions of the presentinvention, descarboxamide derivatives of the corresponding prior artcompounds of FIG. 1. Other such derivatives are also contemplated inconjunction herewith.

FIG. 3 shows, in accordance with this invention, several of anotherclass of preferred compounds, also useful for NOS inhibition.

FIG. 4 provides a generic structural formula of a class of compositionsincluded in this invention, in accordance with various reduced-amidecompounds described elsewhere herein, such a formula relating, but notlimited, to the compounds of FIG. 3 and other compounds provided insubsequent figures, charts and/or tables.

FIG. 5 provides in schematic format a proposed model for binding of theretro-inverso-dipeptide amides 3 and 4 (prior art) at the active site ofnNOS such model as can be used as described herein.

FIG. 6, in schematic format, provides another proposed model for bindingof the retro-inverso-dipeptide amide 2 at the active site of nNOS.

FIG. 7 shows, without limitation, various substituted phenyl, reducedamide bond peptidomimetic compositions, in accordance with thisinvention.

FIG. 8 shows, without limitation, a number of representative pyridinyl,reduced amide bond peptidomimetic compositions, also in accordance withthis invention.

FIG. 9 shows several inhibition patterns, consistent with resultsavailable through use of the present invention; A: Dixon plot ofcompetitive inhibition of nNOS by composition 26; and B: Cornish-Bowdenplot of competitive inhibition.

FIG. 10 shows, without limitation, various argininyl and/orpeptidomimetic compositions, substituted in accordance with thisinvention for conformational restriction and/or control. Consistent withbroader aspects of this invention, several structures provided thereinreflect, without limitation, several of numerous representativesubstituents and possible isomeric and/or diastereomeric configurations.

FIGS. 11-16 represent, without limitation, synthetic routes of the sortavailable for several compositions provided in FIG. 10 and/or elsewhereherein. Several such representative compositions show use of variousprotecting group strategies and/or compositions isolated as acid salts,use of which are also in accordance with this invention.

FIG. 17 compares NOS activities of several 4-amino proline coupleddipeptide derivatives, such derivatives examples only of various othercompositions in accordance with this invention.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide peptidomimetic compositions and/or related methods for their usein the inhibition of nitric oxide synthase, thereby addressing variousissues and concerns of the prior art, including those outlined above. Itwill be understood by those skilled in the art that one or more aspectsof this invention can meet certain objectives, while one or more otheraspects can meet certain other objectives. Each objective may not applyequally, in all its respects, to every aspect of this invention. Assuch, the following objects can be viewed in the alternative withrespect to any one aspect of this invention.

It is an object of the present invention to provide a bioisosteric modelfor the design of a wide variety of nitric oxide synthase inhibitors.

It is an object of the present invention to provide peptidomimetic,argininyl and/or guanidinyl compositions, which can be used selectivelyor otherwise in the inhibition of one or more NOS isoforms.

It can also be an object of the present invention, alone or inconjunction with any other objective, to provide an NOS inhibitorcomposition incorporating one or more amine nitrogen centers, which canbe utilized in conjunction with enzyme active site interaction.

It can also be an object of the present invention, alone or inconjunction with any other objective, to provide an NOS inhibitorcomposition with a reduced amide bond, as can be utilized to enhance thestability of such compositions towards in vivo endogenous peptidases.

It can also be an object of the present invention to providecompositions, such as those described herein, designed with a degree ofconformational control so as to maintain and/or enhance desiredselective inhibition.

It can also be an object of the present invention to provide a method ofusing dipeptide and analogous structures of the type illustrated hereinto determine spatial orientation with respect to an NOS isoform andcorrelate observed activity/selectivity to such structure ororientation.

It can also be an object of the present invention, more specifically, toprovide such dipeptides and/or structurally-related analogues, includingproline moieties and the use thereof to affect NOS activity.

It can also be an object of the present invention to provide a method ofusing proline substitution and associated stereo- and regiochemistry toaffect dipeptide structure, conformation and resulting enzymaticinteraction.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and its descriptions ofvarious preferred embodiments, and will be readily apparent to thoseskilled in the art having knowledge of nitric oxide synthase inhibition.Such objects, features, benefits and advantages will be apparent fromthe above as taken in conjunction with the accompanying examples, data,figures and all reasonable inferences to be drawn therefrom.

In part, the present invention includes an N-nitroarginine-containingdipeptide composition as can be represented by a structural formula ofthe type provided in claim 1, below, as described elsewhere hereinand/or consistent with structures provided in one or more of theaccompanying figures. Without limitation, R₁ can include but is notlimited to hydrogen, alkyl, and a prolinyl (2-carboxypyrrolidinyl and/ora, below) moiety. Such alkyl and/or prolinyl moieties can include one ormore substituents of the type described herein or as could becontemplated by those skilled in the art made aware of this invention,such substitutes as can reflect reagents used and/or choice or design ofsynthetic pathway. R₂ can include but is not limited to amino, alkyl,aminoalkyl, aminoprolinamide (or 2-carbamoylpyrrolidinyl and/or b, c andd below) moiety and a diaminocyclopentanecarboxamide (ordiaminoacetamidocyclopentanyl moiety and/or e and f, below) moiety.Likewise, such R₂ moieties can be substituted as referenced above,described herein or as would otherwise be known to those skilled in theart made aware of this invention, such substituents including but notlimited to hydroxy and azido groups.

As described elsewhere herein, these and other compositions of thepresent invention can be provided, depending upon synthetic method,choice of reagents and/or end use application as, without limitation,one of any number of salts, hydrates, solvates, stereoisomers,diastereomers and/or mixtures thereof. In particular, such a compositioncan be a hydrate, a salt and/or a mixture of stereoisomers. In variouspreferred compositions, R₁ is either hydrogen or a 4-aminoprolinylmoiety.

In part, the present invention can also include a dipeptide compositionhaving an N-nitroarginine residue coupled with an amide bond, at aterminus of the nitroarginine residue, to an amino-substituted prolineresidue. Such a composition, as would be represented in the art, has aformula of either Arg^(NO2)-Pro^(NH2), where the substituted prolineresidue is coupled to the N-terminus of the nitroarginine residue, orPro^(NH2)-Arg^(NO2)-NH₂, where the substituted proline residue iscoupled to the C-terminus of the nitroarginine residue. In preferredembodiments, the proline residue is coupled to the N-terminus of thenitroarginine residue, the latter of which has a stereochemicalconfiguration corresponding to the D-isomer, the L-isomer or a mixturethereof. Highly preferred embodiments of such compositions, as supportedby their inhibition activities, have a nitroarginine residue with anL-configuration, and a proline residue with a 4-amino substituent and atrans-diastereomeric configuration.

Accordingly, the present invention further includes a method of using apro line residue to affect nitric oxide synthase inhibition. Such amethod includes (1) providing a dipeptide composition having anN-nitroarginine residue coupled with an amide bond, at a terminus of thenitroarginine residue, to an amino-substituted proline residue, such adipeptide composition as described above or represented by one or moreof the structural formula provided herein; and (2) interacting thedipeptide composition with an isoform of nitric oxide synthase, suchcomposition in an amount and/or interaction at a time sufficient, undereffective conditions, to inhibit nitric oxide synthase activity on anavailable arginine substrate. In preferred embodiments of such a method,the dipeptide composition can be one of the dipeptides described above.In highly preferred embodiments, the proline residue has a 4-aminosubstituent, is coupled to the C-terminus of the nitroarginine residue,and has a trans-diastereomeric configuration. Such a composition, amongothers described herein, can selectively inhibit the neuronal isoform ofnitric oxide synthase, over the inducible and endothelial isoformsthereof.

In part, the present invention can also include a peptidomimeticN-nitroguanidinyl composition as can be represented by the structuralformula provided in claim 14, consistent with those shown in variousfigures and/or as otherwise described herein. Therein, R₁ is XNHR₂,where X is a moiety selected from the group consisting of CH₂ and C(O).R₂ can include but is not limited to a variety of moieties of the typedescribed herein. For instance, in various preferred embodiments, R₂ canbe an alkylamine, wherein the alkyl portion thereof can be methyl, ethylor propyl, but also can include higher homologs and/or their isomers aswould be understood by those skilled in the art. In various otherembodiments, R₂ can be alkylpyridinyl, with the alkyl portion thereofpositioned ortho, meta or para with respect to the heterocyclic nitrogencenter. In preferred such embodiments, the alkyl portion can vary, butis preferably of 1 or 2-carbon length. Alternatively, the pyridine ringsystem (R₂) can be bonded directly to the nitrogen center of R₁. Invarious other embodiments, R₂ can be an alkylphenylalkylamine moiety,where the alkyl portions can vary in carbon length and with positionrelative one to another on the phenyl ring. Representative, non-limitingstructures of such R₂ moieties are as provided, below. As shown anddiscussed elsewhere, moities such as R₂ can, in the broader context ofthis invention, be substituted (e.g. hydroxy, amino, alkyl, etc.) aswould be understood by those skilled in the art made aware of thisinvention, depending on reagent choice and synthetic method usingstraightforward modifications of the techniques described herein.

Regardless, in preferred peptidomimetic compositions, X is a CH₂ moietyand/or R₂ is an alkylamine. Alternatively, where X is a CH₂ moiety, R₂can be one of various other R₂ selections, provided above or asdescribed elsewhere herein.

Alternatively, various preferred embodiments of the present inventioninclude one of numerous peptidomimetic N-nitroguanidinyl compositionshaving the formula shown in FIG. 4. In particular, R can be an alkyl oraromatic/heterocyclic amine. Preferably, but without limitation, R is analkylamine, alkylphenylamine, alkylpyridine, or phenylalkylaminesubstituent, the alkyl moiety of which is methylene, (CH₂)_(n), or ahomolog thereof with n=1-3 (alkylamine), n=0-2 (alkylphenylamine), n=0-2(alkylpyridine), or n=0-2 (phenylalkylamine). Other compositionalembodiments are discussed below, in the context of variousconformational considerations. Such compositions include, withoutlimitation, any salts, hydrates, solvates, prodrugs, metabolites,stereoisomers, diastereomers, isosteres and/or mixtures thereof.

In part, the present invention is also directed to one or more methodsfor the selective inhibition of the neuronal isoform of nitric oxidesynthase-catalyzed production of nitric oxide. Such a method includesthe interaction or binding of one of the present compositions,preferably an N-nitroguanidinyl composition, with an isoform of nitricoxide synthase, particularly neuronal synthase. Compositions useful inconjunction with the present method include those described more fullyabove, such compositions as can be formulated and/or utilized in amountssufficient to inhibit nitric oxide formation via the correspondingisoform, preferably with selective inhibition of the neuronal isoformover the inducible and endothelial isoforms. More particularly, suchcompositions are utilized in amounts sufficient to affect synthaseactivity on arginine substrates. The composition interaction and isoforminhibition can be determined or analyzed by arginine conversion, or thedecrease thereof, and measured by various spectroscopic and/or assaytechniques.

More specifically, the present invention also includes a method of usingreduced amide argininyl compositions to inhibit isoforms of nitric oxidesynthase. Such compositions include those discussed more fully, above.The interaction or binding thereof with an isoform of nitric oxidesynthase, in the presence of one or more co-factors of the sortdescribed herein, serve to inhibit production of nitric oxide. As withother methods of this invention, effective levels or quantities of suchcompounds will be readily determined by those skilled in the art madeaware of this invention, as will techniques and procedures for theiruse.

Illustrating the design and development of several preferred embodimentsof this invention, peptidomimetic modifications were made on prior artcompounds 1-3. (FIG. 1) Huang, H; Martasek, P.; Roman, L. J.; Silverman,R. B. Synthesis and Evaluation of Peptidomimetics as SelectiveInhibitors and Active Site Probes of Nitric Oxide Synthase. J. Med.Chem. 2000, 43, 2938-45. Incorporation of protecting groups at theN-terminus of the dipeptide and masking of the NH— group of the peptidebond resulted in a dramatic loss in potency of nNOS, demonstratingimportance of the α-amino group of the dipeptide and NH-moiety of thepeptide bond for binding at the enzyme active site. Removal of thecarboxamide group (compositions 4-6, FIG. 2), as one modificationassociated with this invention, had an effect (see Table 1) on bothpotency and selectivity.

Of the many possible bioisosteric modifications of the amino or NHmoieties, the reduced amide bond (—CH₂—NH—) was considered.Representative compounds 7-9 (FIG. 3) contain features beneficial toinhibition of NOS: First, several amine nitrogen centers, useful forinteraction with the enzyme active site; and second, a lack of amidebonding for in vivo stability toward endogenous peptidases.

With reference to FIG. 3 and examples 1-8, reduced amide bond analogues(7-9) were synthesized according to the methods of Scheme 1, usingWeinreb amide 10 as an intermediate. Nahm, S.; Weinreb, S. M.N-Methoxy-N-methylamides as effective acylating agents. TetrahedronLett. 1981, 22, 3815-8. The Weinreb amide was reduced to an aldehydeusing lithium aluminum hydride according to a modified procedure of Goelet al. Goel, O. P.; Krolls, U.; Stier, M.; Kesten, S.N-tert-Butoxycarbonyl-L-Leucinal. Organic Syntheses 1988, 67, 68-71. Theresulting N-Boc-nitro-L-argininal (11) and mono Boc-protectedalkanediamines (12) were reductively coupled using sodiumtriacetoxyborohydride in dry methanol, providing the reduced dipeptides13. Tamura, S. Y.; Semple, J. E.; Ardecky, R. J. Novel and GeneralMethod for the Preparation of Peptidyl Arginals. Tetrahedron Lett. 1996,37, 4109-12. Graham, S. L.; deSolms, S. J.; Guiliani, E. A.; Kohl, N.E.; Mosser, S. D.; Oliff, A. L.; Pompliano, D. L.; Rands, E.; Breslin,M. H.; Deana, A. A.; Garsky, V. M.; Scholz, T. H.; Gibbs, J. B.; Smith,R. L. Pseudpeptide Inhibitors of Ras Farnesyl-Protein Transferase. J.Med Chem. 1994, 37, 725-32. After purification of thee compounds,cleavage of the Boc groups was achieved with TFA. Compositions 7-9 wereisolated as pale yellow powders after lyophilization, and the elementalanalyses showed that all of these compounds were triple trifluoroaceticacid dihydrate salts. As described elsewhere herein, analogousprocedures can be used to prepare various other reduced amide bondcompositions.

The K_(i) data for the reduced amide bond analogues (7-9) are given inTable 1, below, along with the data for the corresponding dipeptides(1-3) and descarboxamide analogues (4-6). Reduction of the carbonylgroup of the amide bond either preserves or improves the potency towardnNOS. Compound 7 shows the best potency over nNOS (K_(i)=120 nM) as wellas the highest selectivity over eNOS (>2500-fold) and iNOS (320-fold) inthese series of compounds. The length of the amine side chain seems tohave only a minor effect on the potency for all isoforms of NOS; 8 and 9inhibit nNOS with K_(i) values of 290 nM and 460 nM, respectively.However, the shorter chain has better potency as well as selectivity.TABLE 1 NOS Inhibition by the Reduced-Amide Bond Analogues 7-9 andN^(ω)- Nitroarginine-Containing Dipeptides (1-6)^(a) K_(i) (μm)^(b)Selectivity^(c) NNOS INOS ENOS eNOS/nNOS iNOS/nNOS 1^(d) 0.13 25 2001538 192 2^(d) 0.33 97 245 742 294 3^(d) 0.45 104 141 313 231 4^(e) 0.54100 199 368 185 5^(e) 0.46 118 213 463 256 6^(e) 0.35 108 70 200 308 70.12 39 314 2617 325 8 0.29 73 524 1807 252 9 0.46 123 411 893 267With reference to Table 1,^(a)the enzymes used for the K_(i) measurements are recombinant ratnNOS, recombinant murine iNOS, and recombinant bovine eNOS.^(b)The K_(i) values represent at least duplicate measurements; standarddeviations of ±8-12% were observed.^(c)The ratio of K_(i) (eNOS or iNOS) to K_(i)(nNOS); all arenNOS-selective.^(d)Data taken from Huang, H; Martasek, P.; Roman, L. J.; Masters, B. S.S.; Silverman, R. B. N N^(ω)-Nitroarginine-Containing Dipeptide Amides.Potent and Highly Selective Inhibitors of Neuronal Nitric OxideSynthase. J. Med Chem. 1999, 42, 3147-53.^(e)Data taken from Huang, H; Martasek, P.; Roman, L. J.; Silverman, R.B. Synthesis and Evaluation of Peptidomimetics as Selective Inhibitorsand Active Site Probes of Nitric Oxide Synthase. J. Med. Chem. 2000, 43,2938-45.

Comparing the data for compositions 7-9 with those for 4-6, to examinethe intrinsic effect of deletion of the amide carbonyl group, thepotency on nNOS and iNOS are about the same or increased, but thepotency with eNOS has greatly decreased. In particular, the largeincrease in selectivity for nNOS over eNOS by 7 appears related to a4.5-fold increase in potency for nNOS and a 1.5-fold decrease in potencyfor eNOS. For 8 and 9 this selectivity increase is believed driven moreby large decreases in potency for eNOS (2.5-fold and almost 6-fold,respectively). The selectivity for nNOS over eNOS is significantlyincreased for 7-9, perhaps with the implication that the carbonyl moietyof the amide bond might not be necessary for its activity toward nNOSand iNOS, but that the rigid —CO—NH— group interacts better with theactive site of eNOS than the reduced, flexible —CH₂—NH— group. Thedifference may also be the result of the nonbasic amide nitrogenbecoming basic when reduced to the corresponding amine, which may notbind as well to eNOS. Regardless, the data demonstrates that reducedamide bond peptidomimetics of this invention, as illustrated bycompositions 7-9, are significant surrogates for the dipeptideinhibitors of nNOS.

Libraries of nitroarginine-containing dipeptides, dipeptide esters, anddipeptide amides have been synthesized as possible candidates fornNOS-inhibition based on two observations. First, the prior artL-nitroarginine (L-NA) itself is a potent and selective nNOS-inhibitor(K_(i)=15 nM for nNOS, and 39 nM for eNOS) and has about 250-foldselectivity in favor of nNOS over iNOS. Second, as mentioned above,prior art L-arginine-containing dipeptides are good substrates for thevarious isoforms of NOS, suggesting that the active site of NOS isflexible enough to accommodate larger molecules than arginine. Indeed,crystal structures of the oxygenase domains of eNOS and iNOS show thereis a large opening, which allows the diffusion of both the substrate andthe product (L-arginine and L-citrulline). Furthermore, there aresignificant structural differences between isoforms just outside of thesubstrate-binding pocket. From these two aspects, it was hypothesizedthat dipeptides containing nitroarginine could fit into the active siteof NOS, and at the same time utilize the structural differences toachieve isoform selectivity.

Twelve nitroarginine (L-, or D-) containing dipeptides and dipeptideesters were synthesized in the standard way, and 152 dipeptide amideswere prepared using a solid-phase synthesis method, and they were testedagainst each isoform of NOS. Among these libraries of compounds, severaldipeptides were found to be potent and nNOS-selective inhibitors. Thedipeptide amides containing amino acids with a nitrogen-containing sidechain, such as Lys, Orn, Dbu, were relatively potent inhibitors of nNOS.They also have great selectivity over eNOS implying that the terminalamine group is significant for the selectivity over eNOS. Among thedipeptide esters, D-Phe-D-Arg^(NO2)-OMe showed excellent selectivity fornNOS over iNOS (1800-fold), although the potency is weak and theselectivity for nNOS over eNOS is minimal. See U.S. Pat. No. 6,274,557,the entirety of which is incorporated herein by reference.

Without limitation, when L-Arg^(NO2) is at the N-terminus of thedipeptide inhibitors, it appears an L-amino acid is also favored at theC-terminus. But when Arg^(NO2) is at the C-terminus, the amino acid wasmore selective as the D-isomer. From these observations, aretro-inverso-dipeptide model was proposed. In this case, thenitroarginine residue binds to the same binding site, regardless of itsposition in the dipeptide (FIG. 5). For example, D-Arg^(NO2) of(schematic 4 in FIG. 5), may flip over 180° to assume an L-Arg^(NO2)configuration (like schematic 3 in FIG. 5) at the N-terminus for binding(FIG. 5). Molecular modeling and energy minimization of theretro-inverso-dipeptide amides (schematics 3 and 4 in FIG. 5) gaveperfect overlap; furthermore, recent ENDOR spectroscopic resultsconfirmed that the dipeptides bind to holo-nNOS similarly from the pointof view of the nitroguanidino functionality.

If a retro-inverso binding model holds for prior artD-Phe-D-Arg^(NO2)-OMe (2) at the active site of nNOS, then it can beexpected to bind as shown in FIG. 6. An important terminal nitrogeninteraction of schematic 1 (or schematic 3, 4 in FIG. 5) in the activesite is lost; instead the phenyl ring moiety replaces the C-terminalresidue active site: explaining the weaker potency and minimalselectivity for nNOS over eNOS of 2 (FIG. 6). However, the replacementof the phenyl ring could also attribute to the high selectivity of nNOSover iNOS (1800-fold); it would not be expected as effective as theamine group moiety at the nNOS active site, but much more unfavorable inthe active site of iNOS.

Without limitation to any one theory or mode of operation, the presentinvention can be considered in light of the foregoing hypothesis:selectivity for nNOS over eNOS derived from the terminal nitrogen, withselectivity over iNOS from an aromatic ring moiety. Accordingly, thepresent invention can provide a basis for design of numerouscompositions: such as but not limited to nNOS-selective inhibitorshaving an aromatic ring and terminal nitrogen moieties within adipeptide-like structure. As discussed above, it was also contemplatedthat a successful surrogate of the amide bond, namely, the reduced-amidebond, could be incorporated into such a structure. Based on suchconsiderations and observations, the present invention further includesvarious heterocyclic or aromatic, reduced-amide bond peptidomimeticcompounds as selective inhibitors of nNOS. (See FIGS. 7 and 8, withreference to FIGS. 3 and 4.) TABLE 2 NOS Inhibition by the aromatic,reduced-amide analogs.^(a)

K_(i)(μM)^(b) Selectivity^(c) Compound nNOS INOS eNOS iNOS/nNOSENOS/nNOS D-Phe-D-Arg^(NO2)-OMe (2)^(d) 2 3600 5 1800 2.5 RedAm-Ethyl(59) 0.12 39 314 320 2577 15 (A, n = 0, m = 0, m-) 2.20 279 78.6 127 3616 (A, n = 0, m = 0, p-) 2.28 339 116 149 51 17 (A, n = 1, m = 0, o-)20.3 86.3 86.1 4.25 4.24 18 (A, n = 1, m = 0, m-) 2.06 572 185 278 90 19(A, n = 1, m = 0, p-) 1.60 326 303 204 189 20 (A, n = 2, m = 0, o-) 1.36184 30.1 135 22.1 21 (A, n = 2, m = 0, m-) 1.36 166 47.6 122 35 22 (A, n= 2, m = 0, p-) 0.74 339 170 458 230 23 (A, n = 0, m = 1, o-) 5.55 29.339.8 5.28 7.17 24 (A, n = 0, m = 1, m-) 0.21 80 194 381 924 25 (A, n =0, m = 1, p-) 1.37 335 409 245 299 26 (A, n = 0, m = 2, o-) 0.05 3.51105 70.2 2121 27 (A, n = 0, m = 2, m-) 2.21 260 100 118 45.2 28 (A, n =0, m = 2, p-) 1.66 360 414 217 249 29 (B, n = 1, o-) 2.90 154 443 53 15330 (B, n = 1, m-) 0.55 141 523 256 951 31 (B, n = 1, p-) 1.59 392 589247 370 32 (B, n = 2, o-) 2.21 72.6 123 33 254 33 (B, n = 2, m-) 2.06178 275 86 133 34 (B, n = 2, p-) 0.76 395 193 520 56

With reference to Table 2, ^(a)the K_(i) values are calculated from themeasured IC₅₀ values and the enzymes used are recombinant rat nNOS,recombinant murine iNOS, and recombinant bovine eNOS. ^(b)The K_(i)values represent at least duplicate measurements; standard deviations of±8-12% were observed. ^(c)The ratio of K_(i) (eNOS or iNOS) to K_(i)(nNOS); all are nNOS selective. ^(d)Data taken from ref 10.

With reference to examples 10-38, below, compounds (14-34) were preparedby reductive amination, using N-Boc-L-nitroargininal (35) as the keyintermediate and it was coupled with various aromatic diamines.

Compounds 17-22 were synthesized according to Scheme 2.N-Boc-L-nitroargininal (26) was coupled with (2-, 3-,4-aminomethyl)anilines (36-38), and [2-,3-,4-(2-aminoethyl)]anilines(39-41), respectively. The reductive amination was performed in decentyield without protection of the aniline group because alkyl amines aremuch more reactive than aniline.

Most of the corresponding aromatic diamines were commercially available,but 2- and 3-(2-aminoethyl)aniline (39, 40) were synthesized (Scheme 3).The closest starting materials were the corresponding(nitrophenyl)acetonitriles, which were sequentially reduced by BH₃-THFcomplex and tin chloride-hydrate to give 39 and 40.

The aniline amino group of the same (aminoalkyl)anilines (36-41) alsowere coupled with N-Boc-L-nitroargininal (Scheme 4). In this case,protection of the alkylamine moieties preceded the reductive amination(except 45 and 48 were commercially available). Using two equivalents of(aminoalkyl)aniline was enough for mono protection, while eightequivalents was necessary for the monoprotection of α,ω-dialkylamine inthe reference.

Among these target molecules, 14 (FIG. 7) could not be made using thesame synthetic method. The reductive coupling of mono-Boc-protectedbenzene-1,2-diamine (44) with N-Boc-L-nitroargininal was not successful,and the bulky Boc-group was thought to be the reason. However it turnedout that the low reactivity of benzene-1,2-diamine, not the stericfactor, was the cause, because the reductive aminations with either freebenzene-1,2-diamine or mono-methylcarbamated benzene-1,2-diamine provedto be fruitless. Furthermore, N-Boc-L-nitroargininal is also somewhatunreactive, since it exists as two forms; cyclized hemiaminal andaldehyde in solution. To accomplish this reaction, different reactionconditions; other than reductive amination are needed. But furtherinvestigation was postponed until enzyme tests show that the series ispotent enough to warrant synthesis of this analogue.

Scheme 5 shows the synthetic route for the pyridyl, reduced-amide bondanalogs. All of the aminoalkyl pyridines (53-58) were commerciallyavailable, and in each case the reductive amination withN-Boc-L-nitroargininal worked well.

All products from the coupling reactions were purified by columnchromatography using various solvent systems, and the Boc group wasdeprotected in 30% TFA/methylene chloride. They showed >90% purity byHPLC. All twenty compounds were tested on each isozyme of NOS todetermine IC₅₀ values. All of the aromatic, reduced-amide analogues arecompetitive inhibitors of the three isoforms of NOS. Representativeplots for the competitive inhibition of nNOS by 26 are shown in FIG. 9.Dixon analysis and the method of Cornish and Bowden were used todetermine the type of inhibition. The K_(i) data for the aromatic,reduced-amide bond analogues (15-34) are given in Table 2 along with thedata for the prior art D-Phe-D-Arg^(NO2)-OMe and the reduced-amide ethyl(RedAm-ethyl) analog (59).

As shown in the Table 2, all tested aromatic, reduced-amide bondanalogues as representative of this invention, were found to benNOS-selective inhibitors. When compared to the non-aromatic compound,RedAm-ethyl (59), the potencies on nNOS are decreased (except 26), butmost of compounds showed still higher potency than the prior artD-Phe-D-Arg^(NO2)-OMe. The potencies with iNOS were also decreased fromthose of 59 except for 23 and 26, but not as low as the aforementionedprior art compound. However, the K_(i) value with iNOS is closer to thatof 59 or prior art L-Arg^(NO2)-L-Dbu-NH₂ rather than toD-Phe-D-Arg^(NO2)-OMe, indicating that a desired unfavorable interactionof the aromatic moiety in the iNOS active site did not occur in thisseries.

The first two benzenediamine compounds (15, 16) were found to be lesssatisfactory inhibitors of nNOS and iNOS than the RedAm-ethyl (59),while having little difference toward eNOS inhibition. This results inthe dramatic decrease of isoform selectivity of nNOS over eNOS (from2577-fold to 36 and 5 1-fold). This implies the amino residue of theC-terminal phenyl ring is not long enough for an effective interactionwith the tetrahydrobiopterin. Further, these two compounds would not bepreferred drug candidates because they were found to be unstable in theHepes buffer (pH 7.5), the medium for the enzyme assay.

The (aminomethy)laniline series (17-19) are also less preferred asinhibitors of nNOS and iNOS. The rigid C-terminal aniline structure doesnot seem to be favored for the interaction with H₄B in the nNOS activesite. This is clear in the case of the o-isomers (17, 23), which showedalmost the same potency with all of the isozymes, thereby resulting inthe loss of isoform selectivity. However, when the (aminomethyl)anilinemoiety was attached at the anilineamino group (but the same lengh,23-25), a much better result was obtained for the m-isomer (24),indicating that a flexible amino residue is better for selectiveinteraction with nNOS; also, spatial geometry appears to be aconsideration. Compound 24 showed almost the same potency andselectivity as RedAm-ethyl (59) implying that the spatial geometry inthe enzyme active site is similar.

The inhibitory potency of the (aminoethyl)aniline series 20-22 and 26-28also varied depending on the location of the rigid phenyl ring. Itappears that the p-isomer is preferred when in phenyl ring is at theC-terminus (22), but the o-isomer is much more preferred when theflexible alkylamine residue is at the C-terminus (26) in this length ofcompounds. Compound 26 was the best nNOS inhibitor (K_(i)=0.05 μM) ofall of the compounds this series, and the isoform selectivity over eNOSwas also excellent (>2000-fold); however the selectivity over iNOS isonly 70-fold.

The variation of the inhibitory potencies with nNOS as a result of asmall structural change was also found in the pyridinyl series (29-34).While they are generally weak inhibitors of nNOS; 30 was the best ofthese compounds, with good isoform selectivity over iNOS and eNOS. Whilethe predicted increase in isoform selectivity of nNOS over iNOS was notfully realized in the aromatic, reduced-amide bond peptidomimeticcompositions, a high potency with nNOS as well as high selectivity ofnNOS over eNOS as retained in some of these compounds (24, 26, 30), aswell as good selectivity over iNOS.

Yet another consideration germane to this invention is steric and/orconformational control of the inhibitor compositions. With reference tothe prior art, the dipeptide L-Are^(NO2)-L-Dbu-NH₂, the L-Dbu fragmenthas free rotation with respect to the aminoethyl side chain.Determination of the exact spatial position of the amino group at theenzyme is difficult, and also there might be a loss of activity and/orselectivity because of conformational flexibility. Accordingly,L-Arg^(NO2)-L-Dbu-NH² can also be used as a point for development ofconformationally restricted dipeptide derivatives and/or relatedpeptidomimetic compositions having a degree of conformational control,in accordance with this invention.

Accordingly, in part, the present invention includes numerous dipeptidecompositions, including but not limited to those shown in FIGS. 10-17.Such compositions include, without limitation, any possible salts,hydrates, solvates, prodrugs, metabolites, stereoisomers, diastereomers,isosteres and/or mixtures thereof, as would be understood by thoseskilled in the art.

More specifically, the present invention also includes a method of usingproline substitution of a dipeptide structure to affect and/or inhibitnitric oxide synthase activity. Such proline-substituted compositionsinclude those discussed more fully herein. The interaction or bindingthereof with an isoform of nitric oxide synthase, optionally in thepresence of one or more co-factors of the sort described herein, serveto inhibit production of nitric oxide. As with other methods of thisinvention, effective levels or quantities of such compositions will bereadily determined by those skilled in the art made aware of thisinvention as will various techniques and procedures for their use.

In accordance therewith, dipeptides and/or peptidomimetic compositionsof this invention have also been synthesized using an exocyclic nitrogenon a proline derivative to form the dipeptide bond. Such structuresprovide rigid analogs of the aforementioned prior art compounds butwithout a substituted peptide bond. Some of these compositions were themost potent of any described herein (see FIG. 17).

Trans derivatives were more potent than cis derivatives in these series;4N-(L-Arg^(NO2))-L-trans-Pro^(4NH2)-NH₂ 71 is the most potent compoundwith an IC₅₀ of 0.5 μM. This compound represents the firstconformationally restricted analog of L-Arg^(NO2)-L-Dbu-NH₂, with asimilar IC₅₀ and selectivity over iNOS. L-Arg^(NO2)-containingdipeptides were more potent than D-Arg^(NO2) analogs. The diastereomer,4N-(L-Arg^(NO2))-D-trans-Pro^(4NH2)-NH₂, 100 showed a greaterselectivity over iNOS. D-Arg^(NO2)-containing dipeptides showed IC₅₀higher than 100 μM. Intriguingly, L-trans-4-aminoproline-containingdipeptides are the most potent compounds in all the series (seecompounds 69 and 87 in Tables 3 and 4, in conjunction with severalexamples, below.). This indicates which conformation ofL-Arg^(NO2)-L-Dbu-NH₂ interacts with the enzyme.

Trans derivatives were purified by HPLC affording 210 mg of the mostpotent compound 4N(L-Arg^(NO2))-L-trans-Pro^(4NH2)-NH_(2b) 71, 10 mg of4N-(D-Arg^(NO2))-L-trans-Pro^(4NH2)-NH₂ 99, 30 mg of4N-(D-Arg^(NO2))-D-trans-Pro^(4NH2)-NH₂ 101, and 180 mg of4N-(L-Arg^(NO2))-D-trans-Pro^(4NH2)-NH₂ 100. Compound 74 has beenassayed against nNOS (IC₅₀=0.73 μM) and iNOS (IC₅₀=13.5 μM). Thiscompound is less selective than 71 against iNOS but their potenciesagainst nNOS are similar. The cis isomer of compound 74 showed anIC₅₀=100 μM against nNOS and an IC₅₀ higher than 100 μM against iNOS.This result clearly stresses the importance of the trans configurationon the conformationally restricted analogs of L-Arg^(NO2)-L-Dbu-NH₂. Forinstance, the diastereomer mixture of the compound 78 showed an IC₅₀against nNOS higher than 100 μM.

The present invention also provides a pharmaceutical compositioncomprising a dipeptide or peptidomimetic composition of this inventionin conjunction with a physiologically or otherwise suitable formulation.In a preferred embodiment, the present invention includes one or moreNOS inhibitors as set forth above formulated into compositions togetherwith one or more non-toxic physiologically tolerable or acceptablediluents, carriers, adjuvants or vehicles that are collectively referredto herein as diluents, for parenteral injection, for oral administrationin solid or liquid form, for rectal or topical administration, or thelike. The resulting compositions can be, in conjunction with the variousmethods described herein, administered to humans and animals eitherorally, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, locally, or as a buccal or nasal spray.

Compositions suitable for parenteral administration can comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into such sterile solutions or dispersions. Examples ofsuitable diluents include water, ethanol, polyols, suitable mixturesthereof, vegetable oils and injectable organic esters such as ethyloleate. Proper fluidity can be maintained, for example, by the sue of acoating such a lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants.

Compositions can also contain adjuvants such as preserving, wetting,emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be insured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin. Besides such inert diluents, the composition can also includesweetening, flavoring and perfuming agents. Suspensions, in addition tothe active compounds, may contain suspending agents, as for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonit,agar—agar and tragacanth, or mixtures of these substances, and the like.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compositions and/or related methods of thepresent invention, including the use of reductive amination and/or theresulting guanidinyl compositions, the aforementioned descarboxamidecompositions, and those prepared to afford or examine conformationalcontrol—all as are available through the synthetic methodologiesdescribed herein. In comparison with the prior art, the presentcompositions and related methods provide results and data which aresuprising, unexpected, and contrary to the prior art. While the utilityof this invention is illustrated through the use of several compositionsand methods which can be used therewith, it will be understood by thoseskilled in the art that comparable results are obtainable with variousother compositions and/or their methods of use, as are commensurate withthe scope of this invention.

General Methods. NOS assays were recorded on a Perkin-Elmer Lambda 10UV/vis spectrophotometer. ¹H NMR spectra were recorded on a Varian Inova500-MHz NMR spectrometer. Chemical shifts are reported as δ values inparts per million downfield from TMS (δ 0.00) as the internal standardin CDCl₃. For samples run in D₂O, the HOD resonance was arbitrarily setat 4.80 ppm. An Orion Research model 701 pH meter with a generalcombination electrode was used for pH measurements. Electrospray massspectra were obtained on a Micromass Quattro II spectrometer. Elementalanalyses were obtained by Oneida Research Services, Inc., Whiteboro,N.Y. Thin-layer chromatography was carried out on E. Merck precoatedsilica gel 60 F₂₅₄ plates. Amino acids were visualized with a ninhydrinspray reagent or a UV/vis lamp. E. Merck silica gel 60 (230-400 mesh)was used for flash column chromatography.

High-performance liquid chromatography was performed on a Beckman SystemGold (Model 125P solvent module and Model 166 detector). In case ofanalytical HPLC, samples were analyzed by elution from a Hypersil ODSC₁₈ column (Agilent, 5 μm, 4.0×250 mm), with a flow rate of 1 mL/min.The mobile phase was a gradient from 100% solvent A (0.1% TFA in water)and 0% solvent B (0.1% of TFA in CH₃CN) to 60% solvent A and 40% solventB over 5 min, then to 30% solvent A and 70% solvent B over 20 min and to100% solvent A over 10 min. Sample elution was detected by absorbance at254 nm. For the purification of final products, a Whatman Partsil C₁₈semi-prep HPLC column (9.4×125 mm) was used. Samples were eluted using agradient of 100% solvent A (0.1% TFA in water) to 70% of solvent B (0.1%of TFA in CH₃CN) over 30 min at a flow rate of 4 mL/min.

Reagents and Materials. Amino acids were purchased from AdvancedChemTech, Inc. NADPH, calmodulin, and human ferrous hemoglobin wereobtained from Sigma Chemical Co. Tetrahydrobiopterin (H₄B) was purchasedfrom Alexis Biochemicals. HEPES, DTT and conventional organic solventswere purchased from Fisher Scientific. 2,3,4-(aminomethyl) aniline waspurchased from TCI, America. All other chemicals were purchased fromAldrich, unless otherwise stated.

Enzyme and Assay. All of the NOS isoforms used were recombinant enzymesoverexpressed in E. coli from different sources; there is very highsequence identity for the isoforms from different sources. The murinemacrophage iNOS was expressed and isolated according to the procedure ofHevel et al. Hevel, J. M.; White, K. A.; Marletta, M. A Purification ofthe Inducible Murine Macrophage Nitric Oxide Synthase. J. Biol. Chem.1991, 266, 22789-91. The rat nNOS was expressed (Roman, L. J.; Sheta, E.A.; Martasek, P.; Gross, S. S.; Liu, Q.; Masters, B. S. S. High LevelExpression of Functional Rat Neuronal Nitric Oxide Synthase inEscherichia coli. Proc. Natl. Acad. Sci. USA. 1995, 92, 8428-32) andpurified as described. Martasek, P.; Liu, Q.; Roman, L. J; Gross, S. S.;Sessa, W. C.; Masters. B. S. S. Characterization of Bovine EndothelialNitric Oxide Synthase Expressed in Escherichia coli. Biochem, Biophys.Res. Commun. 1996, 219, 359-65. The bovine eNOS was isolated asreported. Martasek. P.; Liu, Q.; Roman, L. J.; Gross, S. S.; Sessa, W.C.; Masters B. S. S. Characterization of Bovine Endothelial Nitric OxideSynthase Expressed in Escherichia coli. Biochem. Biophys. Res. Commun.1996, 219, 359-65. Nitric oxide formation from NOS was monitored by thehemoglobin capture assay as described. Hevel, J. M.; Marletta, M. ANitric Oxide Synthase Assays. Methods Enzymol. 1994, 133, 250-8.

With respect to examples 4-8, a typical assay mixture for nNOS contained10 μM arginine, 1.6 mM CaCl₂, 11.6 μg/ml calmodulin, 100 μMdithiotheitol, 100 μM NADPH, 6.5 μM tetrahydrobiopterin, and 3 mMoxyhemoglobin in 100 mM Hepes buffer (pH 7.5). The mixture for the iNOSassay included 10 μM arginine, 100 ,μM dithiotheitol, 100 μM NADPH, 6.5μM tetrahydrobiopterin, and 3 mM oxyhemoglobin in 100 mM Hepes buffer(pH 7.5). The assay mixture for eNOS contained 80 μM oxyhemoglobin, 10μM arginine, 100 μM dithiotheitol, 10 μM CaCl₂, 1 μg/ml calmodulin, 5 μMtetrahydrobiopterin, and 100 μM NADPH in 50 mM Hepes buffer (pH 7.5).All assays were in a final volume of 600 μL and were initiated withenzyme. Nitric oxide reacts with oxyHb to yield methemoglobin which wasdetected at 401 nm (ε=19700 M⁻¹cm⁻¹).on a Perkin-Elmer Lambda 10 U_(vis)spectrophotometer.

With, respect to examples 10-38, a typical assay mixture for nNOScontained 3-15 μM L-arginine, 1.6 mM CaCl₂, 11.6 μg/mL calmodulin, 100μM DTT, 100 μM NADPH, 6.5 μM BH₄, and 3 mM oxyhemoglobin in 100 mM Hepes(pH 7.5). The reaction mixture for the iNOS assay included 10 μML-arginine, 100 μM DTT, 100 μM NADPH, 6.5 μM BH₄, and 3 mM oxyhemoglobinin 100 mM Hepes (pH 7.5). The eNOS assay mixture contained 3-25 μML-arginine, 10 μM CaCl₂, 1 μg/mL calmodulin, 100 μM DTT, 100 μM NADPH, 5μM BH₄, and 80 μM oxyhemoglobin in 50 mM Hepes (pH 7.5). All assays werein a final volume of 600 μL and were initiated by enzyme. Nitric oxidereacts with oxyHb to yield methemoglobin which was detected at 401 nm(ε=19700 M⁻¹cm⁻¹) on a Perkin-Elmer Lambda 10 UV/vis spectrophotometer.

Inhibition Methods. The reversible inhibition of NOS was studied underinitial rate conditions with the hemoglobin assay as described above.The apparent K_(i) values were obtained by measuring percent inhibitionin the presence of 10 μM L-arginine with at least three concentrationsof inhibitor. The parameters of the following inhibition equation werefitted to the initial velocity data: % Inhibition=100[I]/{[I]+K_(i)(1+[S]/K_(m))}. K_(m) values for L-arginine were 1.3 μM (nNOS), 8.2 μM(iNOS), and 1.7 μM (eNOS). The selectivity of an inhibitor was definedas the inverse ratio of the respective K_(i) values.

Example 1

N^(α)-(tert-Butoxycarbonyl)-L-nitroarginine N-methyl-O-methylcarboxamide(10). This compound was prepared from 12.8 g (40.1 mmol) ofN^(α)-(tert-butoxycarbonyl)-L-nitroarginine as described in thereference below, except that isobutyl chloroformate was used instead ofmethyl chloroformate. The residue was further evacuated on an oil pumpto give a white solid product (12.9 g, 89%): ¹H NMR δ5.65 (d, 1H, N—H,J=9.0), 4.69 (t, 1H, J=9.0), 3.79 and 3.73 (s, 3H), 3.25 and 3.10 (s,3H), 3.64 (m, 1H), 3.32 (m, 1H), 1.79 (m, 2H), 1.63 (m, 2H), 1.47 (s,9H). Goel, O. P.; Krolls, U.; Stier, M.; Kesten, S.N-tert-Butoxycarbonyl-L-leucinal. Org. Synth. 1988, 67, 68-71.

Example 2

N^(α)-(tert-Butoxycarbonyl)-L-nitroargininal (11). This compound wasprepared according to the method in the reference below. From 3.62 g of10 (10 mmol), the white powder product 2.17 g (72%) was obtained, and itwas stored in a deep freezer (−80° C.) prior to use. ¹H NMR showed that11 is a mixture of the free aldehyde and cyclized hemiaminal. Tamura, S.Y.; Semple, J. E.; Ardecky, R. J. Novel and General Method for thePreparation of Peptidyl Arginals. Tetrahedron Lett. 1996, 37, 4109-12.

Example 3

(4S)-4-N-tert-Butoxycarbonylamino-5-(2-[N-tert-butoxycarbonylaminoethyl]aminopentyl]-N′-nitroguanidine (13a). To a solution of 11(303 mg, 1 mmol) in dry methanol,N^(α)-tert-butoxycarbonyl-1,2-ethanediamine (12, n=1; 184 μl, 1 mmol)and 3 Å molecular sieves were added and stirred at room temperature.After being stirred for 1 h, the reaction mixture was treated withsodium triacetoxyborohydride (334.6 mg, 1.5 mmol) and stirred overnight.The reaction mixture was filtered, and the filtrate was concentrated invacuo. The residue was purified by flash column chromatography on silicagel (EtOAc:MeOH=1:1) to afford 13a (201 mg, 45%) as a pale yellow solid:¹H NMR (500 MHz, CD₃OD) δ3.67 (m, 1H), 3.33 (m, 2H), 3.04-3.20 (m, 2H),2.62-2.73 (m, 4H), 1.52-1.75 (m, 4H), 1.45 (brs, 18H).

Example 4

(4S)-4-N-tert-Butoxycarbonylamino-5-(2-[N-tert-butoxycarbonylaminopropyl]aminopentyl]-N′-nitroguanidine (13b). This compound (234 mg,51%) was prepared as described above usingN^(α)-tert-butoxycarbonyl-1,3-propanediamine (12b, n=2): 1H NMR (500MHz, CDCl₃) δ4.90 (m, 1H), 3.69 (m, 1H), 3.18-3.21 (m, 4H), 2.77(m, 2H),2.64 (m, 2H), 1.50-1.65 (m, 6H), 1.44 (brs, 18H).

Example 5

(4S)-4-N-tert-Butoxycarbonylamino-5-(2-[N-tert-butoxycarbonylaminobutyl]aminopentyl]-N′-nitroguanidine (13c). This compound (265 mg,56%) was prepared as described above usingN′-tert-butoxycarbonyl-1,4-butanediamine: ¹H NMR (500 MHz, CDCl₃) δ4.67(m, 1H), 3.71 (m, 1H), 3.14 (m, 4H), 2.72 (t, 2H, J=6.0), 2.64 (m, 2H),1.71 (m, 2H), 1.49-1.54 (m, 8H), 1.45 (brs, 18H).

Example 6

(4S)—N-(4-Amino-5-[aminoethyl]aminopentyl)-N′-nitroguanidine (7).Compound 13a (201 mg, 0.45 mmol) was treated with 10 mL oftrifluoroacetic acid/CH₂Cl₂ (1:1 v/v) for 30 min. Excess TFA and solventwere removed by evaporation. The residue was dissolved in a small amountof water, which was washed with ether and lyophilized to give a paleyellow foam (110 mg, 99%): ¹H NMR (500 MHz, D₂O) δ3.63 (m, 1H),3.32-3.39 (m, 6H), 3.24 (m, 2H), 1.69-1.78 (m, 4H). HRMS (M+1) calcd forC₈H₂₁N₇O₂ 248.183, found 248.180. Anal (C₈H₂₁N₇O₂.3TFA.2H₂O) C, H, N.

Example 7

(4S)—N-(4-Amino-5-[aminopropyl]aminopentyl)-N′-nitroguanidine (8). Thiscompound was prepared as described above using compound 13b: ¹H NMR (500MHz, D₂O) δ3.62 (m, 1H), 3.32 (m, 2H), 3.24 (m, 2H), 3.13 (m, 2H), 3.01(m, 2H), 2.03 (quin, 2H, J=7.0), 1.69-1.78 (m, 4H). HRMS (M+1) calcd forC₉H₂₃N₇O₂ 262.199, found 262.195. Anal (C₉H₂₃N₇O₂.3TFA.2H₂O) C, H, N.

Example 8

(4S)—N-(4-Amino-5-[aminobutyl]aminopentyl)-N—-nitroguanidine (9). Thiscompound was prepared as described above using compound 13c: ¹H NMR (500MHz, D₂O) δ3.75 (m, 1H), 3.62 (m, 2H), 3.32 (m, 2H), 3.24 (m, 2H), 2.94(m, 2H), 1.67-1.82 (m, 8H). HRMS (M+1) calcd for C₁₀H₂₅N₇O₂: 276.214,found 276.214. Anal (C₁₀H₂₅N₇O₂.3TFA.2H₂O) C, H, N.

Example 9

Determination of K_(i) Values. The apparent K_(i) values were obtainedby measuring percent inhibition in the presence of 10 μM L-arginine withat least three concentrations of inhibitor. The parameters of thefollowing inhibition equation (Segel, I. H. Enzyme Kinetics; John Wileyand Sons; New York, 1975; p105) were fitted to the initial velocitydata: % inhibition=100[I]/{[I]+K_(i)(1+[S]/K_(m))}. K_(m) values forL-arginine were 1.3 μM (nNOS), 8.2 μM (iNOS), and 1.7 μM (eNOS). Theselectivity of an inhibitor was defined as the ratio of the respectiveK_(i) values.

Example 10

General procedure for the Reductive amination of 15-34. To a solution ofN^(□)-(tert-butoxycarbonyl)-L-nitroargininal (1 equiv.) in dry methanol,aromatic diamine (1.5 equiv.) and 3 Å molecular sieves were added andstirred at the room temperature. After being stirred for 1 h, thereaction mixture was treated with sodium triacetoxyborohydride (2equiv.) and was stirred for overnight. The reaction mixture was filteredand the filtrate was concentrated in vacuo. The residue was purified byflash column chromatography on silica gel to afford the ÑBoc product asa solid, which was treated with 30% TFA in CH₂Cl₂ for 2 h. Excesstrifluoroacetic acid and solvent were removed by evaporation. Theresidue was dissolved in a small amount of water, which was washed withether and lyophilized.

Example 11

N-(4S)-{[4-Amino-5-(3-amino)phenylamino]pentyl}-N′-nitroguanidine (15).This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (500 mg, 1.66 mmol) and(3-aminophenyl)carbamic acid tert-butyl ester (514.9 mg, 2.49 mmol). Thepurification by flash column chromatography on silica gel(EtOAc:hexane=3:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{[4-amino-5-(3-amino)phenylamino]pentyl}-N′-nitroguanidine(300 mg, 37%). Removal of the Boc-group yielded 170 mg of 6 (95%, purplesolid): ¹H NMR (500 MHz, D₂O) δ7.11 (t, J=8 Hz, 1H), 6.60 (d, J=8 Hz,1H), 6.52 (d, J=8 Hz, 1H), 6.47 (s, 1H), 3.31 (m, 1H), 3.22 (m, 2H),3.10 (brs, 2H), 1.41-1.73 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd forC₁₂H₂₂N₇O₂ 296.1835, found 296.1846.

Example 12

N-(4S)-{[4-Amino-5-(4-amino)phenylamino]-pentyl}-N′-nitroguanidine (16).This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (500 mg, 1.66 mmol) and(4-aminophenyl)carbamic acid tert-butyl ester (515 mg, 2.49 mmol). Thepurification by flash column chromatography on silica gel(EtOAc:hexane=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{[4-amino-5-(4-amino)phenylamino]pentyl}-N′-nitroguanidine(400 mg, 49%). Removal of Boc-group from 360 mg yielded 197 mg of 7(92%, orange solid): ¹H NMR (500 MHz, D₂O) □7.04(d, J=8 Hz, 2H), 6.65(d, J=8 Hz, 2H), 3.34 (m, 1H), 3.30 (m, 2H), 3.14 (brs, 2H), 1.40-1.81(m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₂H₂₂N₇O₂ 296.1835, found296.1848.

Example 13

N-(4S)-[4-Amino-5-(2-aminobenzylamino)pentyl]-N′-nitroguanidine (17).This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (359 mg, 1.19 mmol),(2-aminomethyl)aniline (204 μl, 2.49 mmol). The residue was purified byflash column chromatography on silica gel (CH₂Cl₂:MeOH=3:2) to affordN^(□)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-amino)benzylamino]-pentyl}-N′-nitroguanidine(270 mg, 56%) as a white solid. Removal of the Boc-group (30% TFA) gavea yellow foam (204 mg, 95% yield): ¹H NMR (400 MHz, D₂O) δ7.57 (m, 2H),7.46 (d, J=3.6 Hz, 1H), 7.29 (m, 1H), 3.44 (m, 1H), 3.31 (m, 2H), 3.17(m, 2H), 1.46-1.90 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₃H₂₃N₇O₂310.1991, found 310.1920.

Example 14

N-(4S)-[4-Amino-5-(3-aminobenzylamino)pentyl]-N′-nitroguanidine (18).This compound was prepared as described above usingN^(□)-(tert-butoxycarbonyl)-L-nitroargininal (500 mg, 1.66 mmol),(3-aminomethyl)aniline (284 μl, 2.49 mmol). The residue was purified byflash column chromatography on silica gel (CH₂Cl₂:MeOH=3:2) to affordN^(□)-(tert-butoxycarbonyl)-(4S)-[4-amino-5-(2-aminobenzylamino)pentyl]-N′-nitroguanidine(400 mg, 58.9%) as a white solid. Removal of Boc-group gave a lightbrown foam of 9 (278 mg, 92% yield): ¹H NMR (500 MHz, D₂O) δ7.23(m, 2H),7.18 (brs, 1H), 7.13 (d, J=3 Hz, 1H), 4.01 (s, 2H), 3.39 (q, J=6 Hz,1H), 3.12 (d, J=5.5 Hz, 2H), 2.93 (brs, 2H), 1.25-1.65 (m, 4H). HRMS(ES) (m/z): M+H⁺ calcd for C₁₃H₂₃N₇O₂ 310.1991, found 3 10.2008.

Example 15

N-(4S)-[4-Amino-5-(4-aminobenzylamino)pentyl]-N′-nitroguanidine (19).This compound was prepared as described above usingN-(tert-butoxycarbonyl)-L-nitroargininal (500 mg, 1.66 mmol),(4-aminomethyl)aniline (284 μl, 2.49 mmol). The residue was purified byflash column chromatography on silica gel (CH₂Cl₂:MeOH=3:2) to affordN^(α)-(tert-butoxycarbonyl)-(4S)-[4-amino-5-(2-aminobenzylamino)pentyl]-N′-nitroguanidine(410 mg, 62%) as a white solid. An aliquot of 304 mg was treated with30% TFA in CH₂Cl₂ to give 19 (204 mg, 89%, brown solid): ¹H NMR (500MHz, D₂O) δ7.43(d, J=8.5 Hz, 2H), 7.28 (d, J=8.5 Hz, 2H), 4.16 (s, 2H),3.52 (q, J=5.5 Hz, 1H), 3.26 (d, J=5.5 Hz, 2H), 3.10 (brs, 2H),1.45-1.73 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₃H₂₃N₇O₂ 310.1991,found 310.1992.

Example 16

N-(4S)-{[4-Amino-5-(2-aminophenyl)ethylamino]pentyl}-N′-nitroguanidine(20). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (400 mg, 1.32 mmol) and2-(2-aminoethyl)aniline (361 mg, 2.65 mmol). The purification by flashcolumn chromatography on silica gel (CH₂Cl₂:MeOH=1:1) gaveN^(□)-(tert-butoxycarbonyl)-(4S)-{[4-amino-5-(2-aminophenyl)ethylamino]pentyl}-N′-nitroguanidine(415 mg, 74%). Removal of the Boc-group yielded 282 mg of 20 (89%, brownsolid): ¹H NMR (500 MHz, D₂O) δ7.26(m, 4H), 3.53 (m, 1H), 3.26 (m, 4H),3.12 (brs, 2H), 2.95 (t, J=6.8 MHz, 2H), 1.36-1.80 (m, 4H). HRMS (ES)(m/z): M+H⁺ calcd for C₁₄H₂₆N₇O₂ 324.2148, found 324.2151.

Example 17

N-(4S)-{[4-Amino-5-(3-aminophenyl)ethylamino]pentyl}-N′-nitroguanidine(21). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (400 mg, 1.32 mmol) and3-(2-aminoethyl)aniline (361 mg, 2.65 mmol). The purification by flashcolumn chromatography on silica gel (CH₂Cl₂:MeOH=1:1) gaveN^(α)-(tert-Butoxycarbonyl)-(4S)-[4-amino-5-(3-amino-phenyl)-ethylamino)-pentyl]-N′-nitroguanidine(250 mg, 45%). Removal of the Boc-group yielded 189 mg of 21 (99%, brownsolid): ¹H NMR (400 MHz, D₂0) 87.30(d, J=7.2 Hz, 1H), 7.21 (d, J=7.2 Hz,1H), 7.11 (m, 2H), 3.49 (m, 1H), 3.20 (m, 4H), 3.11 (brs, 2H), 2.90 (t,J=7.2 MHz, 2H), 1.33-1.77 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd forC₁₄H₂₆N₇O₂ 324.2148, found 324.2147.

Example 18

N-(4S)-{[4-Amino-5-(4-aminophenyl)ethylamino]pentyl)-N′-nitroguanidine(22). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (500 mg, 1.66 mmol) and4-(2-aminoethyl)aniline (338 μL, 2.49 mmol). The purification by flashcolumn chromatography on silica gel (CH₂Cl₂:MeOH=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-[4-amino-5-(4-aminophenyl)ethylamino)pentyl]-N′-nitroguanidine(380 mg, 54%). Removal of the Boc-group yielded 218 mg of 22 (95%,yellow solid): ¹H NMR (500 MHz, D₂O) δ7.28(d, J=8 Hz, 2H), 7.21 (d, J=8Hz, 2H), 3.53 (m, 1H), 3.24 (m, 4H), 3.15 (brs, 2H), 2.94 (t, J=7.5 MHz,2H), 1.43-1.77 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₄H₂₆N₇O₂324.2148, found 324.2150. Anal Calcd for C₁₄H₂₅N₇O₂.3TFA.H₂O: C, 35.15;H, 4.42; N, 14.35; Found: C, 35.65; H, 4.25; N, 14.30.

Example 19

N-(4S)-{4-Amino-5-[(2-aminomethyl)phenylamino]pentyl}-N′-nitroguanidine(23). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (200 mg, 0.73 mmol) and(2-aminobenzyl)carbamic acid tert-butyl ester (324 mg, 1.46 mmol). Thepurification by flash column chromatography on silica gel(EtOAc:hexane=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(3-aminomethyl)phenylamino]pentyl}-N′-nitroguanidine(160 mg, 43%). Removal of the Boc-group yielded 92.3 mg of 23 (95%,orange solid): ¹H NMR (400 MHz, D₂O) δ7.55(m, 2H), 7.46 (m, 1H), 7.27(m, 1H), 3.63 (m, 1H), 3.31 (m, 2H), 3.23 (m, 4H), 1.40-1.88 (m, 4H).HRMS (ES) (m/z): M+H⁺ calcd for C₁₃H₂₃N₇O₂ 310.1991, found 310.1931.

Example 20

N-(4S)-{4-Amino-5-[(3-aminomethyl)phenylamino]pentyl}-N′-nitroguanidine(24). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (458.5 mg, 1.52 mmol) and(3-aminobenzyl)carbamic acid tert-butyl ester (675 mg, 3.04 mmol). Thepurification by flash column chromatography on silica gel(EtOAc:hexane=1:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(3-aminomethyl)phenylamino]pentyl}-N′-nitroguanidine(320 mg, 41%). Removal of the Boc-group yielded 120 mg of 24 (62%, brownsolid): ¹H NMR (400 MHz, D₂O) δ7.18(t, J=7.5 Hz, 1H), 6.79 (m, 3H), 3.91(s, 2H), 3.55 (m, 1H), 3.37 (m, 2H), 3.09 (m, 2H), 1.36-1.82 (m, 4H).HRMS (ES) (m/z): M+H⁺ calcd for C₁₃H₂₃N₇O₂ 310.1991, found 310.1992.Anal Calcd for C₁₃H₂₃N₇O₂.3TFA.2H₂O: C, 33.19; H, 4.40, N, 14.26; Found:C, 33.62; H; 4.38; N, 14.19.

Example 21

N-(⁴S)-{4-Amino-5-[(4-aminomethyl)phenylamino]pentyl}-N′-nitroguanidine(25). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (500 mg, 1.66 mmol) and(4-aminobenzyl)carbamic acid tert-butyl ester (569 mg, 2.49 mmol). Thepurification by flash column chromatography on silica gel(EtOAc:hexane=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(4-aminomethyl)phenylamino]pentyl}-N′-nitroguanidine(390 mg, 46%). Removal of the Boc-group yielded 234 mg of 25 (99%, paleyellow solid): ¹H NMR (400 MHz, D₂O) δ7.12(d, J=8 Hz, 2H), 6.65 (d, J=8Hz, 2H), 3.89 (s, 2H), 3.36 (m, 1H), 3.29 (m, 2H), 3.14 (brs, 2H),1.36-1.62 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₃H₂₃N₇O₂ 310.1991,found 310.1992.

Example 22

N-(4S)-{4-Amino-5-[2-(2-aminoethyl)phenylamino]-pentyl}-N′-nitroguanidine(26). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (463.5 mg, 1.53 mmol) and[2-(2-aminophenyl)ethyl]carbamic acid tert-butyl ester (544 mg, 2.30mmol). The purification by flash column chromatography on silica gel(EtOAc:hexane=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[2-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguanidine(300 mg, 38%). Removal of the Boc-group yielded 176 mg of 26 (95%, brownsolid): ¹H NMR (400 MHz, D₂O) δ7.06(t, J=6.8 Hz, 1H), 6.94 (d, J=6.8 Hz,1H), 6.60 (m, 2H), 3.37 (m, 1H), 3.26 (m, 2H), 3.10 (brs, 2H), 3.04 (t,J=7.2 Hz, 2H), 2.72 (t, J=7.2 Hz, 2H), 1.38-1.78 (m, 4H). HRMS (ES)(m/z): M+H⁺ calcd for C₁₄H₂₆N₇O₂ 324.2148, found 324.2137. Anal Calcdfor C₁₄H₂₅N₇O₂.3TFA.H₂O: C, 35.15; H, 4.42; N, 14.35; Found: C, 35.59;H, 4.32; N, 14.41.

Example 23

N-(4S)-{4-Amino-5-[3-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguanidine(27). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (433 mg, 1.43 mmol) and[3-(2-aminophenyl)ethyl]carbamic acid tert-butyl ester (508 mg, 2.15mmol). The purification by flash column chromatography on silica gel(EtOAc:hexane=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[3-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguanidine(314 mg, 42%). Removal of the Boc-group yielded 174 mg of 27 (90%, brownsolid): ¹H NMR (400 MHz, D₂O) δ7.07(t, J=7.6 Hz, 1H), 6.59 (m, 3H), 3.55(m, 1H), 3.45 (m, 2H), 3.31 (m, 2H), 3.09 (m, 4H), 1.34-1.74 (m, 4H).HRMS (ES) (m/z): M+H⁺ calcd for C₁₄H₂₆N₇O₂ 324.2148, found 324.2137.

Example 24

N-(4S)-{4-Amino-5-[4-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguanidine(28). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (477 mg, 1.58 mmol) and[4-(2-amino-phenyl)ethyl]carbamic acid tert-butyl ester (560 mg, 2.37mmol). The purification by flash column chromatography on silica gel(EtOAc:hexane=2:1) gaveN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[4-(2-aminoethyl)phenylamino]pentyl}-N′-nitroguanidine(300 mg, 36%). Removal of the Boc-group yielded 176 mg of 28 (95%, lightbrown solid): ¹H NMR (500 MHz, D₂0) □7.01(d, J=8 Hz, 2H), 6.64 (d, J=8Hz, 2H), 3.60 (m, 1H), 3.49 (m, 1H), 3.29(m, 3H), 3.15 (m, 2H), 2.72 (t,J=7.5 Hz, 2H), 1.42-1.81 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd forC₁₄H₂₆N₇O₂ 324.2148, found 324.2154.

Example 25

N-(4S)-{4-Amino-5-[(pyridin-2-yl)methyl]aminopentyl}-N′-nitroguanidine(29). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (400 mg, 1.32 mmol),1-(pyridin-2-yl)methylamine (204.8 μL, 1.98 mmol). The residue waspurified by flash column chromatography on silica gel (EtOAc:MeOH=1:1)to affordN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(pyridin-2-ylmethyl)amino]pentyl}-N′-nitroguanidine(340 mg, 65%) as a yellow solid. Removal of the Boc-group gave 29 as apale yellow foam (251 mg, 99%, brown solid): ¹H NMR (500 MHz, D₂O)δ8.64(d, J=5.5 Hz, 1H), 8.37 (t, J=8 Hz, 1H), 7.91 (d, J=8 Hz, 1H), 7.83(t, J=6.5 Hz, 1H), 4.51 (s, 2H), 3.57 (m, 1H), 3.37 (m, 2H), 3.12 (brs,2H), 1.42-1.80 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₂H₂₂N₇O₂296.1385, found 296.1837.

Example 26

N-(4S)-{4-Amino-5-[(pyridin-3-yl)methyl]amino]pentyl}-N′-nitroguanidine(30). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (400 mg, 1.32 mmol),1-(pyridin-3-yl)methylamine (202 μL, 1.98 mmol). The residue waspurified by flash column chromatography on silica gel (EtOAc:MeOH=1:1)to affordN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(pyridin-3-ylmethyl)amino]pentyl}-N′-nitroguanidine(300 mg, 58%) as a yellow solid. Removal of the Boc-group gave 30 as agreenish yellow foam (217 mg, 97%): ¹H NMR (500 MHz, D₂O) δ8.78(brs,1H), 8.68 (d, J=5.5 Hz, 1H), 8.56 (d, J=7.5 Hz, 1H), 7.96 (t, J=7.5 Hz,1H), 4.39 (s, 2H), 3.55 (m, 1H), 3.36 (m, 2H), 3.08 (brs, 2H), 1.39-1.82(m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₂H₂₂N₇O₂ 296.1385, found296.1839. Anal Calcd for C₁₂H₂₁N₇O₂.3TFA.2H₂O: C, 32.10; H, 4.19; N,14.56; Found: C, 31.62; H, 4.16; N, 14.47.

Example 27

N-(4S)-{4-Amino-5-[(pyridin-4-yl)methyl]aminopentyl}-N′-nitroguanidine(31). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (420 mg, 1.39 mmol),1-(pyridin-4-yl)methylamine (211 μL, 2.09 mmol). The residue waspurified by flash column chromatography on silica gel (EtOAc:MeOH=5:2)to affordN-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(pyridin-4-ylmethyl)aminopentyl}-N-nitroguanidine (419 mg, 76%) as a yellow solid. Removal of theBoc-group gave 31 as a orange yellow foam (310 mg, 99%): ¹H NMR (500MHz, D₂O) δ8.72(d, J=6 Hz, 2H), 8.01 (d, J=6 Hz, 2H), 4.50 (s, 2H), 3.62(m, 1H), 3.43 (m, 2H), 3.15 (brs, 2H), 1.46-1.83 (m, 4H). HRMS (ES)(m/z): M+H⁺ calcd for C₁₂H₂₂N₇O₂ 296.1385, found 296.1848.

Example 28

N-(4S)-{4-Amino-5-[(2-pyridin-2-yl)ethyl]aminopentyl}-N′-nitroguanidine(32). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (415 mg, 1.37 mmol),2-(pyridin-2-yl)ethylamine (190 μL, 1.51 mmol). The residue was purifiedby flash column chromatography on silica gel (EtOAc:MeOH=1:1) to affordN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-pyridin-2-yl)ethyl]aminopentyl}-N′-nitroguanidine (400 mg, 71%) as a yellow solid. Removal ofthe Boc-group gave 32 as a light brown foam (296 mg, 98%): ¹H NMR (500MHz, D₂O) δ8.38 (d, J=5.5 Hz, 1H), 8.21 (t, J=8 Hz, 1H), 7.66 (d, J=8Hz, 1H), 7.63 (m, 1H), 3.41 (m, 1H), 3.30 (m, 2H), 3.24 (m, 2H), 3.16(m, 2H), 2.95 (brs, 2H), 1.26-1.62 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcdfor C₁₃H₂₄N₇O₂ 310.1991, found 310.1995.

Example 29

N-(4S)-{4-Amino-5-[(2-pyridin-3-yl)ethyl]aminopentyl}-N′-nitroguanidine(33). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (460 mg, 1.52 mmol),2-(pyridin-3-yl)ethylamine dihydrobromide salt (509 mg) 1.82 mmol). Theresidue was purified by flash column chromatography on silica gel(EtOAc:MeOH=1:1) to affordN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-pyridin-3-yl)ethyl]aminopentyl}-N′-nitroguanidine(404 mg, 65%) as a yellow solid. Removal of the Boc-group gave 33 as apale yellow foam (290 mg, 95%): ¹H NMR (500 MHz, D₂O) δ849 (brs, 1H),8.45 (d, J=4.5 Hz, 1H), 8.30 (d, J=6.5 Hz, 1H), 7.77 (ddd, J=4.5, 6 Hz,1H), 3.46 (m, 1H), 3.25 (m, 2H), 3.20 (m, 2H), 3.07 (m, 2H), 3.02 (brs,2H), 1.35-1.71 (m, 4H). HRMS (ES) (m/z): M+H⁺ calcd for C₁₃H₂₄N₇O₂310.1991, found 310.1995.

Example 30

N-(4S)-{4-Amino-5-[(2-pyridin-4-yl)ethylamino]pentyl}-N′-nitroguanidine(34). This compound was prepared as described above usingN^(α)-(tert-butoxycarbonyl)-L-nitroargininal (400 mg, 1.32 mmol),2-(pyridin-4-yl)ethylamine (249 μL, 1.98 mmol). The residue was purifiedby flash column chromatography on silica gel (EtOAc:MeOH=1:1) to affordN^(α)-(tert-butoxycarbonyl)-(4S)-{4-amino-5-[(2-pyridin-4-yl)ethylamino]pentyl}-N′-nitroguanidine(400 mg, 74%) as a yellow solid. Removal of the Boc-group gave 34 as awhite foam (287 mg, 95%): ¹H NMR (500 MHz, D₂O) δ845 (d, J=6 Hz, 1H),7.73 (d, J=6 Hz, 2H), 3.49 (q, J=6 Hz, 1H), 3.30 (m, 2H), 3.21 (m, 1H),3.15 (t, J=7.5 Hz, 2H), 3.03 (brs, 2H), 1.33-1.76 (m, 4H). HRMS (ES)(m/z): M+H⁺ calcd for C₁₃H₂₄N₇O₂ 310.1991, found 310.1994.

Example 31

2-(2-Aminoethyl)aniline (39). To a solution of(2-nitrophenyl)acetonitrile (3 g, 18.5 mmol) was added dropwise a 1 MBH₃-THF solution (125 mL) at 0° C. After 4 h of stirring at 25° C., 6NHCl solution (125 mL) was added to the reaction mixture at 0° C. Afterevaporation of the organic solvent in vacuo, the aqueous phase wasbasified with 4N NaOH solution to pH 10. Then the product was extractedwith EtOAc and the organic phase was dried over MgSO₄ and concentratedin vacuo. A browm liquid (41, 2 g, 10.8 mmol) was isolated. Withoutfurther purification, it was mixed with SnCl₂.2H₂O (13.57 g, 60.2 mmol)and absolute ethanol (20 mL). The suspension was heated at 70° C. undernitrogen. After being stirred for 30 min, the starting materialdisappeared, the solution was allowed to cool, then poured into ice (100g). The pH was made slightly basic (pH 8) by the addition of a 5%aqueous NaHCO₃ solution, and the resulting basic mixture was stirred for1 h. The precipitate was extracted with ethyl acetate (100 mL×3). Theextract was washed with water (50 mL×3) and dried over MgSO₄. Theproduct was obtained in a yield of 52% (1.3 g, brown liquid, over twosteps) after evaporation of the solvent: ¹H NMR (400 MHz, CD₃OD) δ6.97(t, J=7.2 Hz, 2H), 6.72 (d, J=7.2 Hz, 2H), 6.64 (t, J=7.2 Hz, 1H), 2.82(t, J=7.2 Hz, 2H), 2.66 (t, J=7.2 Hz, 2H). HRMS (EI) m/z (M⁺) calcd forC₈H₁₂N₂ 136.1000, found 136.1002.

Example 32

3-(2-Aminoethyl)aniline (40). This compound was prepared as describedabove using (3-nitrophenyl)acetonitrile (2.2 g, 13.6 mmol). Evaporationof the solvent gave the product in a yield of 81% (1.5 g, brown liquid,over two steps): ¹H NMR (400 MHz, CD₃OD) δ7.02 (t, J=7.2 Hz, 1H), 6.59(m, 2H), 6.56 (m, 1H), 2.84 (t, J=7.2 Hz, 2H), 2.64 (t, J=7.2 Hz, 2H).HRMS (EI) m/z (M⁺) calcd for C₈H₁₂N₂ 136.1000, found 136.0980.

Example 33

(3-Aminophenyl)carbamic acid tert-butyl ester (45). A solution ofdi-tert-butyl-dicarbonate (2 g, 9.16 mmol, 1 equiv.) in dioxane (25 mL)was added over a period of 30 min to a solution of 1,3-phenylenediamine(2 g, 18.5 mmol, 2 equiv.) in dioxane (25 mL). The mixture was allowedto stir for 22 h, and the solvent was removed using a rotary evaporator.The residue was purified by flash column chromatography on silica gel(EtOAc:hexane=2:1) to afford 1.7 g of product (89% based on dicarbonate)as a peach color solid: ¹H NMR (500 MHz, CDCl₃) δ7.11 (t, J=8 Hz, 1H),6.60 (d, J=8 Hz, 1H), 6.52 (t, J=8 Hz, 1H), 6.47 (s, 1H), 4.79 (s, 1H),1.44 (s, 9H). HRMS (EI) m/z (M⁺) calcd for C₁₁H₁₆N₂O₂ 208.1211, found208.1201.

Example 34

(2-Aminobenzyl)carbamic acid tert-butyl ester (47). This compound wasprepared as described above using di-tert-butyl-dicarbonate (2 g, 9.16mmol) and 2-aminomethyl-aniline (2 g, 18.5 mmol). The residue waspurified by flash column chromatography on silica gel (EtOAc:hexane=2:1)to afford 1.5 g of product (79% based on dicarbonate) as a light yellowsolid: ¹H NMR (400 MHz, CDCl₃) δ7.10 (t, J=7.6 Hz, 1H), 7.02 (d, J=6.8Hz, 1H), 6.67 (m, 2H), 4.80 (s, 1H), 1.44 (s, 9H). HRMS (EI) m/z (M⁺)calcd for C₁₂H₁₈N₂O₂ 222.1369, found 222.1357.

Example 35

(3-Aminobenzyl)carbamic acid tert-butyl ester (48). This compound wasprepared as described above using di-tert-butyl-dicarbonate (0.89 g,4.07 mmol) and 3-aminomethyl-aniline (1 g, 8.18 mmol). The residue waspurified by flash column chromatography on silica gel (EtOAc:hexane=2:1)to afford 0.95 g of brown liquid (quantitative yield): ¹H NMR (400 MHz,CDCl₃) δ7.08 (t, J=7.6 Hz, 1H), 6.64 (d, J=7.6 Hz, 1H), 6.58 (m, 2H),4.91 (s, 2H), 1.45 (s, 9H). HRMS (EI) m/z (M⁺) calcd for C₁₂H₁₈N₂O₂222.1369, found 222.1357.

Example 36

[(2-Aminophenyl)ethyl]carbamic acid tert-butyl ester (50). This compoundwas prepared as described above using di-tert-butyl-dicarbonate (0.3 g,2.2 mmol) and 2-(2-aminoethyl)aniline (481 mg, 2.2 mmol). The productwas obtained as pale yellow solid in a quantitative yield (544 mg): ¹HNMR (400 MHz, CD₃OD) δ6.97 (t, J=7.2 Hz, 2H), 6.71 (d, J=8 Hz, 1H), 6.63(t, J=7.2 Hz, 1H), 3.18 (t, J=8 Hz, 2H), 2.67 (t, J=8 Hz, 2H), 1.43 (s,9H). HRMS (EI) m/z (M⁺) calcd for C₁₃H₂₀N₂O₂ 236.1524, found 236.1537.

Example 37

[(3-Aminophenyl)ethyl]carbamic acid tert-butyl ester (51). This compoundwas prepared as described above using di-tert-butyl-dicarbonate (401 mg,1.84 mmol) and 3-(2-aminoethyl)aniline (401 mg, 1.84 mmol). The residuewas purified by flash column chromatography on silica gel(EtOAc:hexane=2:1) to afford 508 mg of product (quantitative yield,yellow solid): ¹H NMR (400 MHz, CD₃OD) δ□7.00 (t, J=8 Hz, 1H), 6.58 (m,3H), 3.21 (t, J=7.6 Hz, 2H), 3.63 (t, J=7.6 Hz, 2H), 1.42 (s, 9H). HRMS(EI) m/z (M⁺) calcd for C₁₃H₂₀N₂O₂ 236.1524, found 236.1508.

Example 38

[(4-Aminophenyl)ethyl]carbamic acid tert-butyl ester (52). This compoundwas prepared as described above using di-tert-butyl-dicarbonate (1 g,7.34 mmol) and 4-(2-aminoethyl)aniline (0.8 g, 3.67 mmol). The productwas obtained in a yield of 98% (850 mg, pale yellow solid): ¹H NMR (400MHz, CDCl₃) δ6.96 (d, J=8.4 Hz, 2H), 6.62 (d, J=8.4 Hz, 2H), 3.29 (t,J=6.4 Hz, 2H), 2.66 (t, J=6.4 Hz, 2H), 1.43 (s, 9H). HRMS (EI) m/z (M⁺)calcd for C₁₃H₂₀N₂O₂ 236.1524, found 236.1555.

Example 39

Synthesis of the compositions of examples 40-45 was implemented onsolid-phase. The substituted proline derivatives were prepared followingpublished methodology (Gomez-Vidal, J. A.; Silverman, R. B. Short,Highly Efficient Syntheses of Protected 3-Azido- and 4-Azidoproline andTheir Precursors. Org. Lett. 2001, 3, 2481-2484. b) Gomez-Vidal, J. A.;Forrester, M. T.; Silverman, Richard B. Mild and Selective Sodium AzideMediated Cleavage of p-Nitrobenzoic Esters. Org. Lett. 2001, 3,2477-2479) or obtained from commercial sources. Enzymatic activity wasdemonstrated using techniques and procedures known in the art and/or asotherwise referenced herein. Likewise, standard assay techniques wereused to demonstrate utility of this aspect of the invention.

Example 40 Dipeptide derivatives with 4-azido- and 4-amino-L- andD-proline at the N-terminus

Various compounds in accordance with this example can be obtained asshown in FIG. 11. Rink amide resin was loaded with Fmoc- orBoc-protected L-Arg^(NO2) or D-Arg^(NO2). Fmoc deprotection was carriedout with a 20% piperidine solution in DMF. Boc deprotection was carriedout using a modified Burgess methodology. (Zhang, A. J.; Russell, D. H.;Zhu, J.; Burgess, K. A Method for Removal of N-BOC Protecting Groupsfrom Substrates on TFA-sensitive Resins. Tetrahedron Lett. 1998, 39,7439-7442.) Two equivalents of TBSOTf were used to effect Bocdeprotection at room temperature instead of an excess of TMSOTf. Afurther modification using 2 equiv. of TMSOTf instead of TBSOTf and TBAFwas introduced to avoid the two steps N-Boc deprotection. Parallel solidphase synthesis was carried out to obtain all the stereoisomers of 60from L- or D-Arg^(NO2) and protected L- or D-cis- ortrans-4-azido-proline. Part of the resin 60 was cleaved to obtain allthe stereisomers of compound 61. The azide group in 60 was reduced tothe amino group under Staudinger conditions. (Scriven, E. F. V.;Turnbull, K. Azides: Their Preparation and Synthetic Uses. Chem. Rev.1988, 88, 297-368) All the stereoisomers of compound 62 were obtainedfollowing the synthetic procedure described in FIG. 3. The procedure ofthe example is general and has been followed for the synthesis ofdipeptides derivatives with constrained mimics of L-Dbu at theN-terminal.

Example 41 Dipeptide Derivatives with 4-trifluoroacetamido- and4-amino-L- and D-proline at the C-terminus

The synthesis of dipeptides containing 4-amino proline derivatives atthe C-terminus has been achieved on Rink resin (see FIG. 12).Triethylammonium tris(phenylthio)stannate was used to reduced the azidegroup in 63 to an amino group in 64. This reagent has been described asa very efficient reducing agent with a high chemoselectivity towardazides (Bartra, M.; Romea, P.; Urpi, F.; Vilarrasa, J. A Fast Procedurefor the Reduction of Azides and Nitro Compounds Based on the ReducingAbility of Sn(SR)₃ ⁻ Species. Tetrahedron. 1990, 46, 587-594), and hasbeen applied successfully on solid phase using Rink resin or otherresins. (Kim, J.; Bi, Y., Paikoff, S. J.; Schultz, P. G. The Solid PhaseSynthesis of Oligoureas. Tetrahedron Lett. 1996, 37, 5305-5308.) Kick,E.; Ellman, J. A. Expedient Method for the Solid-Phase Synthesis ofAspartic Acid Protease Inhibitors Directed toward the Generation ofLibraries. J. Med. Chem. 1995, 38, 1427-1430. b) Tortolani, D. R.;Biller, S. A. A Solid Phase Synthesis of Miconazole Analogs via anIodoetherification Reaction. Tetrahedron Lett. 1996, 37, 5687-5690).After azide reduction the resultant amine is protected as atrifluoroacetamide 65 (a) Greene, T. W. and Wuts, P. G. M. ProtectiveGroups in Organic Synthesis; John Wiley & Sons, Inc.: New York, 1999; p556. b) Katritzky, A. R., Yang, B., Semenzin, D.(Trifluoroacetyl)benzotriazole: A Convenient TrifluoroacetylatingReagent. J. Org. Chem. 1997, 62, 726-728). This additional step isnecessary because of the azide instability during N-Boc deprotectionusing TMSOTf This is not a general occurrence because this reaction hasbeen described previously with N-Boc deprotection in the presence of asecondary azide. (Nicolaou, K. C.; Koumbis, A. E.; Takayanagi, M.;Natarajan, S.; Jain, N. F.; Bando, T.; Li, H.; Hughes, R. TotalSynthesis of Vancomycin-Part 3: Synthesis of the Aglycon. Chem. Eur. J.1999, 5, 2622-2647)

The new protecting group should be orthogonal to Boc and stable toTMSOTf. It should also be eliminated using conditions that do not affectthe Rink resin or the stereochemistry of the final dipeptide.(Trifluoroacetyl)benzotriazole has been described as a useful reagentfor the introduction of this protecting group (a) Greene, T. W. andWuts, P. G. M. Protective Groups in Organic Synthesis; John Wiley &Sons, Inc.: New York, 1999; p 556. b) Katritzky, A. R., Yang, B.,Semenzin, D. (Trifluoroacetyl)benzotriazole: A ConvenientTrifluoroacetylating Reagent. J. Org. Chem. 1997, 62, 726-728). Itsdeprotection is accomplished under basic conditions but its stabilitytoward TMSOTf had not been investigated.

The dipeptide 66 was obtained after Boc deprotection and couplingreaction with Boc protected L- or D-Arg^(NO2). Part of the resin 66 wascleaved to obtain dipepetide 67. All the stereisomers of compound 66were obtained using parallel synthesis from the L- or D-cis- ortrans-4-azidoproline and L- or D-Arg^(NO2). Trifluoroacetamide 66 wasdeprotected using LiOH in THF and H₂O to obtain amine 68. This compoundwas cleaved from the resin to obtain dipeptide 69. All the stereoisomersof compound 69 were obtained as described in FIG. 12.

The synthetic procedures and methods of this example proved to besuccessful for the synthesis of various 4-aminoproline containingdipeptides at the C-terminus.

Example 42 Nonnatural Dipeptides Containing L- or D-Arg^(NO2) and3-amino and 4-aminoproline Derivatives

A third family of dipeptides has been synthesized using the exocyclicnitrogen on the proline derivative to obtain the dipeptide bond (seeFIG. 13). These structures can be described as conformationallyrestricted analogs of the lead compound but without a substitutedpeptide bond. Intermediate 64 is coupled to L- or D-Arg^(NO2) to obtaindipeptide 70. This compound was cleaved from the resin to obtain 71. Allthe stereoisomers of 71 were obtained following the scheme of FIG. 13from protected L- and D-cis- and trans-4-aminoproline.

Thirty-two compounds were synthesized following the schemes of FIGS. 11and 12 and eight compounds were synthesized following the scheme of FIG.13. All of these compounds were purified by solid phase extraction(SPE), and the final purity was determined by analytical HPLC and ¹HNMR.A Luna column (Phenomenex, C18-2, 250*4.6 mm, 5 U) was selected as theanalytical column, and a rapid method was developed to analyze eachsample.

Example 43

The synthetic procedure of example 42 was used starting fromN-Boc-L-trans- and cis-4-azidoproline (see FIG. 14). N-Boc-L-Arg^(NO2)was coupled to 72 to obtain dipeptide intermediate 73. This compound wascleaved from the resin to obtain 74. The cis isomer was also obtainedstarting from protected cis-azido-proline.

Example 44 Synthesis of Dipeptides from L-Arg^(NO2) andN-Boc-amino-(3-hydroxycyclopentyl)carboxylic acid orN-Fmoc-amino-(3-hydroxycyclopentyl)carboxylic acid

Rink amide resin was loaded with a racemic mixture ofN-Boc-amino-(3-hydroxycyclopontyl)carboxylic acid using HBTU as acoupling reagent to give intermediate 75 (see FIG. 15). The hydroxygroup in 75 was converted to an azido group under Mitsunobu conditions(Nicolaou, K. C.; Winssinger, N.; Vourloumis, D.; Ohshima, T.; Kim, S.;Pfefferkorn, J.; Xu, J.; Li, T. Solid and Solution Phase Synthesis andBiological Evaluation of Combinatorial Sarcodictyin Libraries. J. Am.Chem. Soc. 1998, 120, 10814-10826) and reduced to an amino group in 76.N-Boc-L-Arg^(NO2) was coupled to give dipeptide 77 that was cleaved toobtain dipeptide 78 as a mixture of 4 diastereomers. This mixture waspre-purified using SPE and two fractions with two differentdiastereomers each were used for enzyme assays.

Example 45

N-Fmoc-amino-(3-hydroxycyclopentyl)carboxylic acid was loaded as aracemic mixture on Rink amide resin to give 79 (see FIG. 16). Thehydroxy group in 79 was converted to an azido group under Mitsunobuconditions and the Fmoc protecting group was removed using 20%piperidine in DMF to give 80. This intermediate was coupled toN-Boc-L-Arg^(NO2) to obtain the dipeptide 81. After azide reductionunder Staudinger conditions the compound was cleaved from the resin togive dipeptide 82 as a mixture of 4 diastereomers. This mixture waspre-purified using SPE before enzyme assays.

Example 46 Enzyme Activity of Dipeptide Analogs Containing ProlineDerivatives at the N- and the C-terminus

Thirty-two derivatives containing proline derivatives at the N- and theC-terminus did not show activity at 100 μM against iNOS (IC₅₀>100 μM).Some compounds have an IC₅₀ value of 100 μM or less against nNOS (seeTable 3 and Table 4). TABLE 3 nNOS activities of the dipeptide analogscontaining proline derivatives at the C-terminus. Activity at 100 μM 67L-Arg^(NO2)-L-trans-Pro^(4NHCOCF3)-NH2 41% 69L-Arg^(NO2)-L-trans-Pro^(4NH2)-NH₂ 50% 83D-Arg^(NO2)-D-cis-pro^(4NH2)-NH₂ 56%

Only three dipeptides analogs containing proline derivatives at theC-terminus exhibited some activity with nNOS at 100 μM (see Table 3above). This supports the importance of a non-substituted peptide bondfor the interaction with the enzyme (Huang, H.; Martasek, P.; Roman, L.J.; Silverman, R. B. Synthesis and evaluation of peptidomimetics asselective inhibitors and active site probes of nitric oxide synthases.J. Med. Chem. 2000, 43, 2938-2945).

Twelve dipeptide analogs containing proline derivatives at theN-terminus showed activity over nNOS at 100 μM (see Table 3).L-trans-Pro^(4NH2)-L-Arg^(NO2)-NH₂ is the most potent compound of theseseries with an IC₅₀ value lower than 50 μM. The azide analog 88 showed athree-fold decrease in activity, which supports an interaction of the4-amino substituent in 87 with the enzyme. The stereochemistry of analog87 is important for its activity. The introduction of D-Arg^(NO2) in 89instead of L-Arg^(NO2) decreased the activity two fold. The inversion ofconfiguration at the Cα position in 87 caused a two fold decreasedactivity (see compound 85). Interestingly, compound 84, containing aD-Arg^(NO2) and a D-proline, showed the same activity as 87 at 100 μM.The activity of the azido dipeptide analog 90 is difficult to explainsince its 4-amino analog 89 is two times less potent. TABLE 4 nNOSactivities of the dipeptide analogs containing proline derivatives atthe N-terminus. Activity @ 100 μM Activity @ 50 μM 62L-cis-Pro^(4NH2)-L-Arg^(NO2)-NH₂ 48% 84 L-cis-Pro^(4NH2)-D-Arg^(NO2)-NH₂24% 43% 85 D-cis-Pro^(4NH2)-L-Arg^(NO2)-NH₂ 42% 86D-cis-Pro^(4NH2)-D-Arg^(NO2)-NH₂ 54% 87L-trans-Pro^(4NH2)-L-Arg^(NO2)-NH₂ 24% 34% 88L-trans-Pro^(4N3)-L-Arg^(NO2)-NH₂ 67% 89L-trans-Pro^(4NH2)-D-Arg^(NO2)-NH₂ 47% 90L-trans-Pro^(4N3)-D-Arg^(NO2)-NH₂ 25% 91D-trans-Pro^(4NH2)-L-Arg^(NO2)-NH₂ 35% 92D-trans-Pro^(4N3)-L-Arg^(NO2)-NH₂ 39% 93D-trans-Pro^(4NH2)-D-Arg^(NO2)-NH₂ 55% 94D-trans-Pro^(4N3)-D-Arg^(NO2)-NH₂ 50%

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are added only by way of example and are not intended tolimit, in any way, the scope of this invention. For instance, numerouscompositions of this invention have been prepared and/or described assalts thereof, but can also be provided as would otherwise be known tothose skilled in the art for purposes of bioavailability or increasedeffectiveness. Likewise, this invention includes various otherN-nitroguanidinyl and/or N-nitroargininyl compounds of the typedescribed herein, such compounds as can be prepared to include a rangeof structural or functional substituents and/or other N- or C-terminussubstituents, as would also be understood by those skilled in the artmade aware of this invention, depending upon choice of reagent(s) orreaction conditions, using known synthetic techniques and/orstraight-forward modifications of the procedures described herein. Otheradvantages, features and benefits will become apparent from the claimsto be filed hereafter, with the scope of such claims determined by theirreasonable equivalents, as would be understood by those skilled in theart.

1. An N-nitroarginine-containing dipeptide composition having thestructural formula

wherein R₁ is selected from the group consisting of hydrogen, alkyl, anda prolinyl moiety; and R₂ is selected from the group consisting ofamino, alkyl, aminoalkyl, an aminoprolinamide moiety, and adiaminocyclopentanecarboxamide moiety.
 2. The composition of claim 1wherein said composition is a hydrate.
 3. The composition of claim 1wherein said composition is a mixture of stereoisomers.
 4. Thecomposition of claim 3 wherein said composition is an acid salt.
 5. Thecomposition of claim 1 wherein R₁ is a 4-aminoprolinyl moiety.
 6. Adipeptide composition comprising an N-nitroarginine (Arg^(NO) ² )residue coupled with an amide bond at a terminus of said nitroarginineresidue to an amino-substituted proline (Pro^(NH) ² ) residue, saidcomposition having a formula selected from the group consisting of:Arg^(NO) ² -Pro^(NH) ² , wherein said substituted proline residue iscoupled to the N-terminus of said nitroarginine residue; and Pro^(NH) ²-Arg^(NO) ² -NH₂, wherein said substituted proline residue is coupled tothe C-terminus of said nitroarginine residue.
 7. The composition ofclaim 6 wherein said formula is Arg^(NO) ² -Pro^(NH) ² , and saidnitroarginine residue has a stereochemical configuration selected fromthe group of consisting of D- and L-isomers.
 8. The composition of claim7 wherein said nitroarginine residue has an L-configuration and saidproline residue has a 4-amino substituent and a trans diastereomericconfiguration.
 9. The composition of claim 8 having a formula selectedfrom the group
 10. A method of using a proline residue to affect nitricoxide synthase inhibition, said method comprising: providing a dipeptidecomposition having an N-nitroarginine residue coupled with an amide bondat a terminus of said nitroarginine residue to an amino-substitutedproline residue; and interacting said dipeptide composition with anisoform of nitric oxide synthase, said composition in an amountsufficient to inhibit nitric oxide synthase activity on an availablearginine substrate.
 11. The method of claim 10 wherein said dipeptidecomposition is selected from the group of compositions of claim
 6. 12.The method of claim 11 wherein said substituted proline residue has a4-amino substituent, is coupled to the C-terminus of said nitroarginineresidue, and has a trans-diastereomeric configuration.
 13. The method ofclaim 12 wherein interaction of said dipeptide composition selectivelyinhibits the neuronal isoform of nitric oxide synthase, over theinducible and endothelial isoforms of nitric oxide synthase.
 14. Apeptidomimetic N-nitroguanidinyl composition having the structuralformula

wherein R₁ is XNHR₂, and X is a moiety selected from the groupconsisting of CH₂ and C(O); and R₂ is selected from the group consistingof CH₂(CH₂)_(n)NH₂, where n=1-3;

where n=0-2; and

where m=0-2 and n=0-2.
 15. The composition of claim 14 wherein X is aCH₂ moiety.
 16. The composition of claim 15 wherein R₂ isCH₂(CH₂),_(n)NH₂ and n=1-3.
 17. The composition of claim 15 wherein R₂is

where n=0-2.
 18. The composition of claim 15 wherein R₂ is

where m=0-2 and n=0-2.
 19. A method of using amide bond reduction toaffect nitric oxide synthase inhibition, said method comprising:providing an N-nitroarginine analog composition with a reduced amidebond, said composition having the structural formula

wherein R₁ is XNHR₂, and X is a moiety selected from the groupconsisting of CH₂ and C(O); and R₂ is selected from the group consistingof CH₂(CH₂)_(n)NH₂, where n=1-3;

where n=0-2; and

where m=0-2 and n=0-2; and interacting said reduced amide analogcomposition with an isoform of nitric acid synthase, said composition inan amount sufficient to inhibit nitric oxide formation.
 20. The methodof claim 19 wherein R₂ is CH₂(CH₂)_(n)NH₂ and n=1-3.
 21. The method ofclaim 19 wherein R₂ is

where n=0-2.
 22. The method of claim 19 wherein R₂ is

where m=0-2 and n=0-2.
 23. The method of claim 19 wherein interaction ofsaid composition selectively inhibits the neuronal isoform of nitricoxide synthase over the inducible and endothelial isoforms of nitricoxide synthase.